U.S. patent application number 14/105646 was filed with the patent office on 2014-04-10 for combination therapy and kit for the prevention and treatment of cystic fibrosis.
This patent application is currently assigned to TRUSTEES OF DARTMOUTH COLLEGE. The applicant listed for this patent is TRUSTEES OF DARTMOUTH COLLEGE. Invention is credited to Prisca Boisguerin, Patrick R. Cushing, Dean R. Madden, Rudolf Volkmer, Lars Vouilleme.
Application Number | 20140100155 14/105646 |
Document ID | / |
Family ID | 50433152 |
Filed Date | 2014-04-10 |
United States Patent
Application |
20140100155 |
Kind Code |
A1 |
Madden; Dean R. ; et
al. |
April 10, 2014 |
COMBINATION THERAPY AND KIT FOR THE PREVENTION AND TREATMENT OF
CYSTIC FIBROSIS
Abstract
A combination therapy and kit including an agent that inhibit
the interaction between CAL and mutant CFTR proteins, in
combination with a CFTR corrector, CFTR potentiator, mucolytic,
anti-inflammatory agent or a combination thereof are provided as is
a method for preventing or treating cystic fibrosis.
Inventors: |
Madden; Dean R.; (Hanover,
NH) ; Cushing; Patrick R.; (Woburn, MA) ;
Boisguerin; Prisca; (Berlin, DE) ; Volkmer;
Rudolf; (Nordwestuckermark, DE) ; Vouilleme;
Lars; (Berlin, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TRUSTEES OF DARTMOUTH COLLEGE |
HANOVER |
NH |
US |
|
|
Assignee: |
TRUSTEES OF DARTMOUTH
COLLEGE
HANOVER
NH
|
Family ID: |
50433152 |
Appl. No.: |
14/105646 |
Filed: |
December 13, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13292151 |
Nov 9, 2011 |
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14105646 |
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13124470 |
Apr 15, 2011 |
8415292 |
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PCT/US2009/061246 |
Oct 20, 2009 |
|
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13292151 |
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61107438 |
Oct 22, 2008 |
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Current U.S.
Class: |
514/1.8 |
Current CPC
Class: |
C07K 7/06 20130101; A61K
45/06 20130101; C07K 14/4703 20130101; C07K 7/08 20130101; A61K
31/47 20130101; A61K 38/08 20130101; C07K 5/1013 20130101; A61K
38/10 20130101; A61K 31/47 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/443 20130101;
A61K 38/04 20130101; A61K 38/08 20130101; A61K 38/16 20130101; A61K
38/04 20130101; A61K 31/443 20130101; A61K 38/16 20130101; A61K
38/10 20130101 |
Class at
Publication: |
514/1.8 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 45/06 20060101 A61K045/06; A61K 38/08 20060101
A61K038/08 |
Goverment Interests
[0002] This invention was made with government support under grant
numbers R01-DK075309, P20-RR018787 and R01 GM-78031 awarded by the
National Institutes of Health. The government has certain rights in
the invention. Work on this invention was also supported by grants
from the Cystic Fibrosis Foundation.
Claims
1. A method for preventing or treating cystic fibrosis comprising
administering to a subject in need of treatment an effective amount
of an agent that selectively inhibits the interaction between a
degradation-prone Cystic Fibrosis Transmembrane Conductance
Regulator (CFTR) and CFTR-Associated Ligand in combination with a
CFTR corrector, CFTR potentiator, mucolytic, anti-inflammatory
agent or a combination thereof, thereby preventing or treating the
subject's cystic fibrosis.
2. The method of claim 1, wherein the degradation-prone CFTR is
.DELTA.F508 CFTR or R1066C CFTR.
3. The method of claim 1, wherein the agent is a peptide or
peptidomimetic.
4. The method of claim 3, wherein the peptide or peptidomimetic is
6 to 20 residues in length.
5. The method of claim 3, wherein the peptide comprises the amino
acid sequence of SEQ ID NO:1, or a derivative thereof.
6. The method of claim 3, wherein the peptide is listed in Table
1.
7. The method of claim 3, wherein the peptide is derivatized with a
label, one or more post-translational modifications, a
cell-penetrating sequence, or a combination thereof.
8. The method of claim 7, wherein the cell-penetrating sequence
comprises an amino acid sequence of SEQ ID NO:42.
9. The method of claim 3, wherein the peptidomimetic is a mimetic
of the amino acid sequence of SEQ ID NO:1.
10. The method of claim 3, wherein the peptidomimetic is listed in
Table 3.
11. A kit comprising (a) an agent that inhibits the interaction
between a degradation-prone Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR) and CFTR-Associated Ligand (CAL); and
(b) a CFTR corrector, CFTR potentiator, mucolytic,
anti-inflammatory agent or a combination thereof.
12. The kit of claim 11, wherein the agent is a peptide or
peptidomimetic.
13. The kit of claim 12, wherein the peptide or peptidomimetic is 6
to 20 residues in length.
14. The kit of claim 12, wherein the peptide comprises the amino
acid sequence of SEQ ID NO:1, or a derivative thereof.
15. The kit of claim 12, wherein the peptide is listed in Table
1.
16. The kit of claim 12, wherein the peptide is derivatized with a
label, one or more post-translational modifications, a
cell-penetrating sequence, or a combination thereof.
17. The kit of claim 16, wherein the cell-penetrating sequence
comprises the amino acid sequence of SEQ ID NO:42.
18. The kit of claim 12, wherein the peptidomimetic is a mimetic of
the amino acid sequence of SEQ ID NO:1.
19. The kit of claim 12, wherein the peptidomimetic is listed in
Table 3.
Description
INTRODUCTION
[0001] This application is a continuation-in-part application of
U.S. patent application Ser. No. 13/292,151, filed Nov. 9, 2011,
which is a continuation-in-part application of U.S. patent
application Ser. No. 13/124,470, filed Apr. 15, 2011, now U.S. Pat.
No. 8,415,292, which claims the benefit of priority of
PCT/US2009/061246, filed Oct. 20, 2009, and U.S. Provisional
Application No. 61/107,438, filed Oct. 22, 2008, which are
incorporated herein by reference in their entireties.
BACKGROUND
[0003] CFTR (Cystic Fibrosis Transmembrane Conductance Regulator)
is the target of mutations that cause cystic fibrosis (CF). CF is
characterized by abnormal endocrine and exocrine gland function. In
CF, unusually thick mucus leads to chronic pulmonary disease and
respiratory infections, insufficient pancreatic and digestive
function, and abnormally concentrated sweat. Seventy percent of the
mutant CFTR alleles in the Caucasian population result from
deletion of phenylalanine at position 508 (.DELTA.F508-CFTR), the
result of a three base pair deletion in the genetic code. Other
mutations have also been described, e.g., a glycine to aspartate
substitution at position 551 (G551D-CFTR) occurs in approximately
3-4% of cystic fibrosis patients.
[0004] The .DELTA.F508-CFTR mutation results in a CFTR protein
capable of conducting chloride, but absent from the plasma membrane
because of aberrant intracellular processing. Under usual
conditions (37.degree. C.), the .DELTA.F508-CFTR protein is
retained in the endoplasmic reticulum (ER), by prolonged
association with the ER chaperones, including calnexin and hsp70.
Over expression of .DELTA.F508-CFTR can result in .DELTA.F508-CFTR
protein appearing at the cell surface, and this protein is
functional once it reaches the cell surface. The .DELTA.F508-CFTR
"trafficking" block is also reversible by incubation of cultured CF
epithelial cells at reduced temperatures (25-27.degree. C.).
Lowered temperature results in the appearance of CFTR protein and
channel activity at the cell surface, suggesting an intrinsic
thermodynamic instability in .DELTA.F508-CFTR at 37.degree. C. that
leads to recognition of the mutant protein by the ER quality
control mechanism, prevents further trafficking, and results in
protein degradation. Chemical chaperones are currently being
developed to restore the folding of .DELTA.F508-CFTR. However, when
.DELTA.F508-CFTR is expressed at the cell-surface following
treatment, CAL (also known as CFTR-associated ligand, PIST, GOPC,
ROS, and FIG) directs the lysosomal degradation of CFTR in a
dose-dependent fashion and reduces the amount of CFTR found at the
cell surface. Conversely, NHERF1 and NHERF2 functionally stabilize
CFTR. Consistent with this role of CAL, RNA interference targeting
of endogenous CAL also increases cell-surface expression of the
disease-associated .DELTA.F508-CFTR mutant and enhances
transepithelial chloride currents in a polarized human patient
bronchial epithelial cell line (Wolde, et al. (2007) J. Biol. Chem.
282:8099-8109).
[0005] Current treatments for cystic fibrosis generally focus on
controlling infection through antibiotic therapy and promoting
mucus clearance by use of postural drainage and chest percussion.
However, even with such treatments, frequent hospitalization is
often required as the disease progresses. New therapies designed to
increase chloride ion conductance in airway epithelial cells have
been proposed, and restoration of the expression of functional CFTR
at the cell surface is considered a major therapeutic goal in the
treatment of cystic fibrosis, a disease that affects .about.30,000
patients in the U.S., and .about.70,000 patients worldwide. For
example, KALYDECO (Ivacaftor; VX-770) is an FDA-approved compound
that `potentiates` the open probability (P.sub.o) of CFTR channels,
including the G551D mutant, and thus ameliorates the underlying
molecular lesion in this group of patients. A 48-week clinical
trial showed excellent efficacy, including a 10.6% improvement in
lung function (predicted forced expiratory volume in 1 second;
FEV1), a 55% drop in pulmonary exacerbations, and a 48 mEq/L
reduction in sweat chloride (Ramsey, et al. (2011) N. Engl. J. Med.
365:1663-72). While showing efficacy in subjects with the G551D
mutation, KALYDECO is not useful as a monotherapy for the largest
group of CF patients. In .about.70% of mutant alleles, Phe508 is
deleted (.DELTA.F508; Kerem, et al. (1989) Science 245: 1073-1080).
As a result, .about.50% of CF patients are .DELTA.F508 homozygous
and .about.40% are heterozygous. Unfortunately, clinical trials in
.DELTA.F508 homozygotes show low efficacy for KALYDECO alone
(Flume, et al. (2012) Chest 142:718-724).
[0006] In the absence of interventions, .DELTA.F508-CFTR exhibits
three defects: folding, gating, and stability (Riordan (2008) Annu.
Rev. Biochem. 77:701-726; Cheng, et al. (1990) Cell 63:827-834;
Lukacs, et al. (1993) J. Biol. Chem. 268:21592-21598; Dalemans, et
al. (1991) Nature 354:526-528). However, if folding is restored,
.DELTA.F508-CFTR retains some channel activity (Drumm, et al.
(1991) Science 254:1797-1799; Denning, et al. (1992) Nature
358:761-764). `Corrector` compounds have been identified such as
corr-4a (Pedemonte et al. (2005) J. Clin. Invest. 115:2564) and
Lumacaftor (VX-809), which partially alleviate the folding defect
and allows some .DELTA.F508-CFTR to reach the apical membrane (Van
Goor, et al. (2009) Pediatr. Pulmonol. 44:S154-S155; Van Goor, et
al. (2011) Proc. Natl. Acad. Sci. USA 108:18843-18848). Although
Lumacaftor yields only limited benefits in monotherapy, it shows
greater efficacy in combination with KALYDECO: 25% of patients
showed a >10% increase in FEV1 and 55% of patients showed >5%
increase, with few adverse effects. While a 5% or 10% improvement
is clinically meaningful, FEV.sub.1 drops approximately 1-2% per
year in CF patients (Dasenbrook, et al. (2008) Am. J. Respir. Crit.
Care Med. 178:814-821; Que, et al. (2006) Thorax 61:155-157), even
in the absence of acceleration by pulmonary exacerbations
(Taylor-Robinson, et al. (2012) Thorax 67:860-866). Thus, further
improvements are required, especially for non-responders and the
40% of .DELTA.F508-CFTR heterozygous patients.
SUMMARY OF THE INVENTION
[0007] The present invention is a method for preventing or treating
cystic fibrosis by administering to a subject in need of treatment
an effective amount of an agent that selectively inhibits the
interaction between a degradation-prone CFTR and CAL in combination
with a CFTR corrector, CFTR potentiator, mucolytic,
anti-inflammatory agent or a combination thereof, thereby
preventing or treating the subject's cystic fibrosis. In one
embodiment, the degradation-prone CFTR is .DELTA.F508 CFTR or
R1066C CFTR. In other embodiments, the agent is a peptide or
peptidomimetic (e.g., 6 to 20 residues in length). In certain
embodiments, the peptide has the amino acid sequence of SEQ ID
NO:1, or a derivative thereof, or is a peptide as listed in Table
1. Moreover, in other embodiments, the peptide is derivatized with
a label, one or more post-translational modifications, a
cell-penetrating sequence (e.g., having an amino acid sequence of
SEQ ID NO:42), or a combination thereof. In still other
embodiments, the peptidomimetic is a mimetic of the amino acid
sequence of SEQ ID NO:1 or is a peptidomimetic as listed in Table
3. A kit containing an agent that inhibits the interaction between
a degradation-prone CFTR and CAL; and a CFTR corrector, CFTR
potentiator, mucolytic, anti-inflammatory agent or a combination
thereof, is also provided.
DETAILED DESCRIPTION OF THE INVENTION
[0008] Novel inhibitors have now been identified that block the
interaction or binding of CFTR with the CAL PDZ binding site by
competitive displacement. By inhibiting this interaction with CAL,
degradation-prone CFTR proteins are stabilized and the amount of
CFTR protein at the cell surface is effectively increased. Indeed,
representative peptide and peptidomimetic CAL inhibitors were shown
to increase the apical cell-surface expression and transepithelial
chloride efflux of the most common CFTR mutation associated with
CF. Accordingly, inhibitors of the present invention find
application in increasing the cell surface expression of
degradation-prone CFTR proteins and in the treatment for CF. In
particular, CAL inhibition is of use in combination therapies for
reversing the .DELTA.F508 stability defect.
[0009] As used herein, "cell surface expression" of a CFTR protein
refers to CFTR protein which has been transported to the surface of
a cell. In this regard, an agent that increases the cell surface
expression of a CFTR protein refers to an agent that increases the
amount of CFTR protein, which is present or detected at the plasma
membrane of a cell, as compared to a cell which is not contacted
with the agent.
[0010] Genetic, biochemical, and cell biological studies have
revealed a complex network of protein-protein interactions that are
required for correct CFTR trafficking, including a number of PDZ
(PSD-95, discs-large, zonula occludens-1) proteins, which act as
adaptor molecules, coupling CFTR to other components of the
trafficking and localization machinery, and to other transmembrane
channels and receptors (Kunzelmann (2001) News Physiol. Sci.
16:167-170; Guggino & Stanton (2006) Nat. Rev. Mol. Cell Biol.
7:426-436). Class I PDZ domains typically recognize C-terminal
binding motifs characterized by the sequence
-(Ser/Thr)-X-.PHI.-COOH (where .PHI. represents a hydrophobic side
chain, and X represents any amino acid) (Harris & Lim (2001) J.
Cell Sci. 114:3219-3231; Brone & Eggermont (2005) Am. J.
Physiol. 288:C20-C29). The cytoplasmic C-terminus of CFTR satisfies
the class I PDZ binding motif, ending in the sequence -Thr-Arg-Leu
(Hall, et al. (1998) Proc. Natl. Acad. Sci. USA 95:8496-8501;
Short, et al. (1998) J. Biol. Chem. 273:19797-19801; Wang, et al.
(1998) FEBS Lett. 427:103-108) and it has been demonstrated that
CFTR C-terminal PDZ-binding motif controls retention of the protein
at the apical membrane and modulates its endocytic recycling
(Moyer, et al. (2000) J. Biol. Chem. 275:27069-27074;
Swiatecka-Urban, et al. (2002) J. Biol. Chem. 277:40099-40105). PDZ
proteins that have been shown to bind or interact with CFTR include
NHERF1 (Na+/H+ exchanger regulatory factor 1; also known as EBP50),
NHERF2 (Na+/H+ exchanger regulatory factor 2, also known as
E3KARP), NHERF3 (Na+/H+ exchanger regulatory factor 3, also known
as CAP70, PDZK1, or NaPi CAP-1), NHERF4 (Na+/H+ exchanger
regulatory factor 4, also known as IKEPP or NaPi CAP-2), and CAL
(CFTR-associated ligand; also known as PIST, GOPC, and FIG; GENBANK
Accession Nos. NP.sub.--065132 and NP.sub.--001017408, incorporated
herein by reference) (Guggino & Stanton (2006) supra; Li &
Naren (2005) Pharmacol. Ther. 108:208-223). Of these proteins, CAL
has been shown to reduce the levels of recombinant wild-type CFTR
found in whole cell lysates and at the cell surface, whereas
overexpression of NHERF1 together with CAL can block this effect on
both wild-type and .DELTA.F508-CFTR (Cheng, et al. (2002) J. Biol.
Chem. 277:3520-3529; Guerra, et al. (2005) J. Biol. Chem.
280:40925-40933). Moreover, RNAi targeting of endogenous CAL
specifically increases cell surface expression of the
.DELTA.F508-CFTR mutant protein and enhances transepithelial
chloride currents in a polarized human patient bronchial epithelial
cell line (Wolde, et al. (2007) J. Biol. Chem. 282:8099-8109).
These data indicate that the PDZ proteins which interact with CFTR
have opposing functions. Thus, targeting the interaction of CAL
with CFTR can stabilize a mutant CFTR protein and facilitate cell
surface expression of the same.
[0011] The CFTR protein and mutants thereof are well-known in the
art and wild-type human CFTR is disclosed in GENBANK Accession No.
NP.sub.--000483, incorporated herein by reference. Misfolding of
mutant CFTR proteins has been shown to dramatically augment the
ubiquitination susceptibility of the protein in post-Golgi
compartments (Swiatecka-Urban, et al. (2005) J. Biol. Chem.
280:36762). Thus, for the purposes of the present invention, the
term "degradation-prone" when used as a modifier of a CFTR protein,
refers to a mutant CFTR protein that exhibits an increased rate of
degradation following initial trafficking to the cell surface and a
decrease in the amount of CFTR protein present at the cell surface
(i.e., plasma membrane). Examples of degradation-prone CFTR
proteins include, but are not limited to .DELTA.F508 CFTR and
.DELTA.70F CFTR (see Sharma, et al. (2004) J. Cell Biol. 164:923).
Other degradation-prone CFTR proteins are known in the art and/or
can be identified by routine experimentation. For example, the rate
or amount of transport of CFTR protein from the cell surface can be
determined by detecting the amount of complex-glycosylated CFTR
protein present at the cell surface, in endoplasmic vesicles and/or
in lysosomes using methods such as cell surface immunoprecipitation
or biotinylation or cell immunocytochemistry with an antibody
specific for CFTR protein. Additional methods, both in vivo and in
vitro, are known in the art that can be used for detecting an
increase or decrease in cell surface expression of a CFTR
protein.
[0012] Because PDZ proteins share overlapping specificities,
particular embodiments of this invention embrace inhibitory agents
that selectively block the interaction or binding between a
degradation-prone CFTR and CAL. As used herein, a "selective
inhibitor of the CFTR and CAL interaction" or "an agent that
selectively inhibits the interaction between the degradation-prone
CFTR and CAL" is any molecular species that is an inhibitor of the
CFTR and CAL interaction but which fails to inhibit, or inhibits to
a substantially lesser degree the interaction between CFTR and
proteins that stabilize degradation-prone CFTR, e.g., NHERF1 AND
NHERF2. Methods for assessing the selectively of an inhibitor of
the CFTR and CAL interaction are disclosed herein and can be
carried out in in vitro or in vivo assays.
[0013] By way of illustration, libraries of agents were screened
for the ability to increase the amount of .DELTA.F508 CFTR at the
apical membrane and to increase the CFTR-mediated chloride efflux
across monolayers of CFBE41O-cells. The magnitude of the functional
rescue of the mutant CFTR protein correlated with the selectivity
of the agent for CAL versus NHERF1 and NHERF2, namely, the more
selective the agent for the CAL binding site, the more effective
the agent was at enhancing chloride efflux. Moreover, upon further
refinement, off-site targets were eliminated by modification of the
amino acid residue at P.sup.-5 (see Example 4).
[0014] Accordingly, the present invention features compositions and
methods for facilitating the cell surface expression of mutant CFTR
by selectively blocking the interaction between a degradation-prone
CFTR and CAL. Agents of the present invention can be any molecular
species, with particular embodiments embracing peptides or mimetics
thereof.
[0015] As used herein, the term "peptide" denotes an amino acid
polymer that is composed of at least two amino acids covalently
linked by an amide bond. Peptides of the present invention are
desirably 6 to 20 residues in length, or more desirably 7 to 15
residues in length. In certain embodiments, a selective inhibitor
of the CFTR and CAL interaction is a 6 to 20 residue peptide
containing the amino acid sequence
Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6 (SEQ ID
NO:1), wherein Xaa.sub.1 is Met, Phe, Leu, Ala or Trp; Xaa.sub.2 is
Gln, Pro, or Phe; Xaa.sub.3 is Ser, Val or Thr; Xaa.sub.4 is Ser or
Thr; Xaa.sub.5 is Lys, Arg or Ile; and Xaa.sub.6 is Ile or Val. In
certain embodiments of the present invention, a selective inhibitor
of the CFTR and CAL interaction is a peptide having an amino acid
sequence as listed in Table 1.
TABLE-US-00001 TABLE 1 SEQ Peptide Designation Peptide Sequence ID
NO: PRC 01 CANGLMQTSKI 2 PRC 02 CGLMQTSKI 3 PRC 03 CFFSTII 4 PRC 04
CFFTSII 5 PRC 05 CMQTSII 6 PRC 06 CMQTSKI 7 PRC 07 CWQTSII 8 PRC 08
CWPTSII 9 PRC 09 CTWQTSII 10 PRC 10 CKWQTSII 11 PRC 11 PHWQTSII 12
PRC 12 FHWQTSII 13 PRC 13 SRWQTSII 14 PRC 17 CANSRWQTSII 15 PRC 25
GLWPTSII 16 PRC 26 SRWPTSII 17 PRC 27 FPWPTSII 18 PRC 30 or
F*-iCal36 *FITC-ANSRWPTSII 19 PRC 36 or iCal36 ANSRWPTSII 20 iCAL42
ANSRLPTSII 21 ANSRAPTSII 22 kCAL01 WQVTRV 23 FITC =
fluorescein.
[0016] In particular embodiments of the present invention, a
selective inhibitor of the CFTR and CAL interaction is a peptide
that binds to CAL, but fails to bind to any other lung epithelial
cell protein containing a PDZ domain including but not limited to
TIP-1, NHERF1 and NHERF2. In accordance with this embodiment, the
inhibitor is "CAL selective." CAL selective inhibitors are
desirably 6 to 20 residue peptide and contain the amino acid
sequence
Xaa.sub.7-Xaa.sub.8-Xaa.sub.9-Xaa.sub.10-Xaa.sub.11-Xaa.sub.12 (SEQ
ID NO:24), wherein Xaa.sub.7 is Met, Phe, Leu, or Ala; Xaa.sub.8 is
Gln, Pro, or Phe; Xaa.sub.9 is Ser, Val or Thr; Xaa.sub.10 is Ser
or Thr; Xaa.sub.11 is Lys, Arg or Ile; and Xaa.sub.12 is Ile or
Val. In specific embodiments, a CAL selective inhibitor is a
peptide of SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22.
[0017] In accordance with the present invention, derivatives of the
peptides of the invention are also provided. As used herein, a
peptide derivative is a molecule which retains the primary amino
acids of the peptide, however, the N-terminus, C-terminus, and/or
one or more of the side chains of the amino acids therein have been
chemically altered or derivatized. Such derivatized peptides
include, for example, naturally occurring amino acid derivatives,
for example, 4-hydroxyproline for proline, 5-hydroxylysine for
lysine, homoserine for serine, ornithine for lysine, and the like.
Other derivatives or modifications include, e.g., a label, such as
fluorescein or tetramethylrhodamine; or one or more
post-translational modifications such as acetylation, amidation,
formylation, hydroxylation, methylation, phosphorylation,
sulfatation, glycosylation, or lipidation. Indeed, certain chemical
modifications, in particular N-terminal glycosylation, have been
shown to increase the stability of peptides in human serum (Powell
et al. (1993) Pharma. Res. 10:1268-1273). Peptide derivatives also
include those with increased membrane permeability obtained by
N-myristoylation (Brand, et al. (1996) Am. J. Physiol. Cell.
Physiol. 270:C1362-C1369). An exemplary peptide derivative is
provided in SEQ ID NO:19 (Table 1).
[0018] In addition, a peptide derivative of the invention can
include a cell-penetrating sequence which facilitates, enhances, or
increases the transmembrane transport or intracellular delivery of
the peptide into a cell. For example, a variety of proteins,
including the HIV-1 Tat transcription factor, Drosophila
Antennapedia transcription factor, as well as the herpes simplex
virus VP22 protein have been shown to facilitate transport of
proteins into the cell (Wadia and Dowdy (2002) Curr. Opin.
Biotechnol. 13:52-56). Further, an arginine-rich peptide (Futaki
(2002) Int. J. Pharm. 245:1-7), a polylysine peptide containing Tat
PTD (Hashida, et al. (2004) Br. J. Cancer 90(6):1252-8), Pep-1
(Deshayes, et al. (2004) Biochemistry 43(6):1449-57) or an HSP70
protein or fragment thereof (WO 00/31113) is suitable for enhancing
intracellular delivery of a peptide or peptidomimetic of the
invention into the cell. Examples of known cell-penetrating
peptides (CPP) are provided in Table 2.
TABLE-US-00002 TABLE 2 CPP Sequence SEQ ID NO: MPG
GALFLGFLGAAGSTMGAWSQPKKKRKV 42 R8 RRRRRRRR 43 Tat (48-60)
GRKKRRQRRRPPQQ 44 Transportan GWTLNSAGYLLGKINLKALAALAKKIL 45 TP10
AGYLLGKINLKALAALAKKIL 46 MAP KLALKLALKALKAALKLA 47 MPG-a
GALFLAFLAAALSLMGLWSQPKKKRKV 48 Penetratin RQIKIWFQNRRMKWKK 49
[0019] Exemplary cell penetrating peptides include WrFKK (SEQ ID
NO:34) and MPG (SEQ ID NO:42).
[0020] While a peptide of the invention can be derivatized with by
one of the above indicated modifications, it is understood that a
peptide of this invention may contain more than one of the above
described modifications within the same peptide.
[0021] As indicated, the present invention also encompasses
peptidomimetics of the peptides disclosed herein. Peptidomimetics
refer to a synthetic chemical compound which has substantially the
same structural and/or functional characteristics of the peptides
of the invention. The mimetic can be entirely composed of
synthetic, non-natural amino acid analogues, or can be a chimeric
molecule including one or more natural peptide amino acids and one
or more non-natural amino acid analogs. The mimetic can also
incorporate any number of natural amino acid conservative
substitutions as long as such substitutions do not destroy the
activity of the mimetic. Routine testing can be used to determine
whether a mimetic has the requisite activity, e.g., that it can
inhibit the interaction between CFTR and CAL. The phrase
"substantially the same," when used in reference to a mimetic or
peptidomimetic, means that the mimetic or peptidomimetic has one or
more activities or functions of the referenced molecule, e.g.,
selective inhibition of the CAL and CFTR interaction.
[0022] There are clear advantages for using a mimetic of a given
peptide. For example, there are considerable cost savings and
improved patient compliance associated with peptidomimetics, since
they can be administered orally compared with parenteral
administration for peptides. Furthermore, peptidomimetics are much
cheaper to produce than peptides.
[0023] Thus, peptides described above have utility in the
development of such small chemical compounds with similar
biological activities and therefore with similar therapeutic
utilities. The techniques of developing peptidomimetics are
conventional. For example, peptide bonds can be replaced by
non-peptide bonds or non-natural amino acids that allow the
peptidomimetic to adopt a similar structure, and therefore
biological activity, to the original peptide. Further modifications
can also be made by replacing chemical groups of the amino acids
with other chemical groups of similar structure. The development of
peptidomimetics can be aided by determining the tertiary structure
of the original peptide, either free or bound to a CAL protein, by
NMR spectroscopy, crystallography and/or computer-aided molecular
modeling. These techniques aid in the development of novel
compositions of higher potency and/or greater bioavailability
and/or greater stability than the original peptide (Dean (1994)
BioEssays 16:683-687; Cohen & Shatzmiller (1993) J. Mol. Graph.
11:166-173; Wiley & Rich (1993) Med. Res. Rev. 13:327-384;
Moore (1994) Trends Pharmacol. Sci. 15:124-129; Hruby (1993)
Biopolymers 33:1073-1082; Bugg, et al. (1993) Sci. Am. 269:92-98).
Once a potential peptidomimetic compound is identified, it may be
synthesized and assayed using an assay described herein or any
other appropriate assay for monitoring cell surface expression of
CFTR.
[0024] It will be readily apparent to one skilled in the art that a
peptidomimetic can be generated from any of the peptides described
herein. It will furthermore be apparent that the peptidomimetics of
this invention can be further used for the development of even more
potent non-peptidic compounds, in addition to their utility as
therapeutic compounds.
[0025] Peptide mimetic compositions can contain any combination of
non-natural structural components, which are typically from three
structural groups: residue linkage groups other than the natural
amide bond ("peptide bond") linkages; non-natural residues in place
of naturally occurring amino acid residues; residues which induce
secondary structural mimicry, i.e., induce or stabilize a secondary
structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix
conformation, and the like; or other changes which confer
resistance to proteolysis. For example, a polypeptide can be
characterized as a mimetic when one or more of the residues are
joined by chemical means other than an amide bond. Individual
peptidomimetic residues can be joined by amide bonds, non-natural
and non-amide chemical bonds other chemical bonds or coupling means
including, for example, glutaraldehyde, N-hydroxysuccinimide
esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide
(DCC) or N,N'-diisopropyl-carbodiimide (DIC). Linking groups
alternative to the amide bond include, for example, ketomethylene
(e.g., --C(C.dbd.O)--CH.sub.2-- for --C(C.dbd.O)--NH--),
aminomethylene (CH.sub.2--NH), ethylene, olefin (CH.dbd.CH), ether
(CH.sub.2--O), thioether (CH.sub.2--S), tetrazole (CN.sub.4--),
thiazole, retroamide, thioamide, or ester (see, e.g., Spatola
(1983) in Chemistry and Biochemistry of Amino Acids, Peptides and
Proteins, 7:267-357, "Peptide and Backbone Modifications," Marcel
Decker, NY).
[0026] As discussed, a peptide can be characterized as a mimetic by
containing one or more non-natural residues in place of a naturally
occurring amino acid residue. Non-natural residues are known in the
art. Particular non-limiting examples of non-natural residues
useful as mimetics of natural amino acid residues are mimetics of
aromatic amino acids include, for example, D- or L-naphylalanine;
D- or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2,
3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- or
L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or
L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;
D-(trifluoromethyl)-phenylglycine;
D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or
L-p-biphenylphenylalanine; D- or
L-p-methoxy-biphenyl-phenylalanine; and D- or
L-2-indole(alkyl)alanines, where alkyl can be substituted or
unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl,
isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino
acid. Aromatic rings of a non-natural amino acid that can be used
in place a natural aromatic ring include, for example, thiazolyl,
thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl,
and pyridyl aromatic rings.
[0027] Cyclic peptides or cyclized residue side chains also
decrease susceptibility of a peptide to proteolysis by
exopeptidases or endopeptidases. Thus, certain embodiments embrace
a peptidomimetic of the peptides disclosed herein, whereby one or
more amino acid residue side chains are cyclized according to
conventional methods.
[0028] Mimetics of acidic amino acids can be generated by
substitution with non-carboxylate amino acids while maintaining a
negative charge; (phosphono)alanine; and sulfated threonine.
Carboxyl side groups (e.g., aspartyl or glutamyl) can also be
selectively modified by reaction with carbodiimides
(R'--N--C--N--R') including, for example,
1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or
1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl or
glutamyl groups can also be converted to asparaginyl and glutaminyl
groups by reaction with ammonium ions.
[0029] Lysine mimetics can be generated (and amino terminal
residues can be altered) by reacting lysinyl with succinic or other
carboxylic acid anhydrides. Lysine and other alpha-amino-containing
residue mimetics can also be generated by reaction with
imidoesters, such as methyl picolinimidate, pyridoxal phosphate,
pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,
O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed
reactions with glyoxylate.
[0030] Methionine mimetics can be generated by reaction with
methionine sulfoxide. Proline mimetics of include, for example,
pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- or
4-methylproline, and 3,3,-dimethylproline.
[0031] One or more residues can also be replaced by an amino acid
(or peptidomimetic residue) of the opposite chirality. Thus, any
amino acid naturally occurring in the L-configuration (which can
also be referred to as R or S, depending upon the structure of the
chemical entity) can be replaced with the same amino acid or a
mimetic, but of the opposite chirality, referred to as the D-amino
acid, but which can additionally be referred to as the R- or
S-form.
[0032] As will be appreciated by one skilled in the art, the
peptidomimetics of the present invention can also include one or
more of the modifications described herein for derivatized
peptides, e.g., a label, one or more post-translational
modifications, or cell-penetrating sequence.
[0033] As with peptides of the invention, peptidomimetics are
desirably 6 to 20 residues in length, or more desirably 7 to 15
residues in length. In certain embodiments, a selective inhibitor
of the CFTR and CAL interaction is a 6 to 20 residue peptidomimetic
based on the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:24. In
certain embodiments of the present invention, a selective inhibitor
of the CFTR and CAL interaction is a peptidomimetic listed in Table
3.
TABLE-US-00003 TABLE 3 Peptide Designation Peptide Sequence SEQ ID
NO: PRC 21 WrFK(K-FITC)-ANSRWPTSII 25 PRC 23 WrFKK-ANSRWPTSII 26
PRC 29 WrFK(K-ROX)-ANSRWPTSII 27 PRC 37 pneaWPTSII 28 B1 fNaRWQTSII
29 B2 fNSRWQTSII 30 B3 knSRWQTSII 31 B4 pnSRWQTSII 32 A6 AnSRWQTSII
33 Lower-case = D-amino acids; FITC = fluorescein; ROX =
6-carboxy-X-rhodamine. Underlined residues indicate cyclized side
chains. WrFKK (SEQ ID NO: 34) is a cell penetrating peptide.
[0034] Also included with the scope of the invention are peptides
and peptidomimetics that are substantially identical to a sequence
set forth herein, in particular SEQ ID NO:1 or SEQ ID NO:24. The
term "substantially identical," when used in reference to a peptide
or peptidomimetic, means that the sequence has at least 75% or more
identity to a reference sequence (e.g., 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%). The length of comparison sequences will generally
be at least 5 amino acids, but typically more, at least 6 to 10, 7
to 15, or 8 to 20 residues. In one aspect, the identity is over a
defined sequence region, e.g., the amino or carboxy terminal 3 to 5
residues.
[0035] The peptides, derivatives and peptidomimetics can be
produced and isolated using any method known in the art. Peptides
can be synthesized, whole or in part, using chemical methods known
in the art (see, e.g., Caruthers (1980) Nucleic Acids Res. Symp.
Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232;
and Banga (1995) Therapeutic Peptides and Proteins, Formulation,
Processing and Delivery Systems, Technomic Publishing Co.,
Lancaster, Pa.). Peptide synthesis can be performed using various
solid-phase techniques (see, e.g., Roberge (1995) Science 269:202;
Merrifield (1997) Methods Enzymol. 289:3-13) and automated
synthesis may be achieved, e.g., using the ABI 431A Peptide
Synthesizer (Perkin Elmer) in accordance with the manufacturer's
instructions.
[0036] Individual synthetic residues and peptides incorporating
mimetics can be synthesized using a variety of procedures and
methodologies known in the art (see, e.g., Organic Syntheses
Collective Volumes, Gilman, et al. (Eds) John Wiley & Sons,
Inc., NY). Peptides and peptide mimetics can also be synthesized
using combinatorial methodologies. Techniques for generating
peptide and peptidomimetic libraries are well-known, and include,
for example, multipin, tea bag, and split-couple-mix techniques
(see, for example, al-Obeidi (1998) Mol. Biotechnol. 9:205-223;
Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997)
Mol. Divers. 3:17-27; and Ostresh (1996) Methods Enzymol.
267:220-234). Modified peptides can be further produced by chemical
modification methods (see, for example, Belousov (1997) Nucleic
Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med.
19:373-380; and Blommers (1994) Biochemistry 33:7886-7896).
[0037] Alternatively, peptides of this invention can be prepared in
recombinant protein systems using polynucleotide sequences encoding
the peptides. By way of illustration, a nucleic acid molecule
encoding a peptide of the invention is introduced into a host cell,
such as bacteria, yeast or mammalian cell, under conditions
suitable for expression of the peptide, and the peptide is purified
or isolated using methods known in the art. See, e.g., Deutscher et
al. (1990) Guide to Protein Purification: Methods in Enzymology
Vol. 182, Academic Press.
[0038] It is contemplated that the peptides and mimetics disclosed
herein can be used as lead compounds for the design and synthesis
of compounds with improved efficacy, clearance, half-lives, and the
like. One approach includes structure-activity relationship (SAR)
analysis (e.g., NMR analysis) to determine specific binding
interactions between the agent and CAL or CFTR to facilitate the
development of more efficacious agents. Agents identified in such
SAR analysis or from agent libraries can then be screened for their
ability to increase cell surface expression of CFTR.
[0039] In this regard, the present invention also relates to a
method for identifying an agent for which facilitates cell surface
expression of a degradation-prone CFTR. The method of the invention
involves contacting CAL with a test agent under conditions allowing
an interaction between the agent and CAL, and determining whether
the agent competitively displaces binding of a degradation-prone
CFTR to CAL. Particular degradation-prone CFTRs that can be used
include, but are not limited to, .DELTA.F508 and R1066C.
[0040] In one embodiment, the method is performed in vivo. Various
detection methods can be employed to determine whether the agent
displaces CFTR from CAL. For example, displacement can be based on
detecting an increase in an amount of CFTR protein on the cell
surface, immunostaining with a specific antibody (e.g., anti-CFTR,
M3A7), or direct visualization (e.g., a CFTR-GFP fusion).
Additional methods useful for determining whether there is an
increase in cell surface protein included cell panning. In cell
panning assays, plates are coated with an antibody that binds to
the cell surface protein. The number of cells that binds to the
antibody coated plate corresponds to an amount of protein on the
cell surface.
[0041] In another embodiment, the method is performed in vitro. In
accordance with this embodiment, a combination of peptide-array
screening and fluorescence polarization is used to identify agents
that bind to an isolated, recombinant CAL PZD domain. For example,
it contemplated that the high-affinity CAL-binding peptides
disclosed herein can be use as reporters for small-molecule
screening assays, wherein the small molecules compete for binding
to the CAL PZD domain. The ability to target PDZ proteins
selectively, using a combination of peptide-array screening and
fluorescence-polarization assays on purified, recombinant PDZ
domains, represents a novel achievement, due to the bi-directional
promiscuity of PDZ:protein interactions. Since PDZ proteins are
implicated in the trafficking and intracellular localization of
many disease-related receptors, selective targeting may provide an
important tool for identifying additional PDZ-based
therapeutics.
[0042] In so far as it is desirable that the agent selectively
inhibit the interaction between CAL and CFTR, a further embodiment
of this invention embraces contacting NHERF1 and/or NHERF2 with an
identified inhibitor of the CAL and CFTR interaction and
determining whether the agent competitively displaces binding to
NHERF1 and/or NHERF2. Agents that fail to inhibit, or inhibit to a
substantially lesser degree the interaction between CFTR and NHERF1
or NHERF2 as compared to CAL, would be considered selective.
[0043] Agents which can be screened in accordance with the methods
disclosed herein can be from any chemical class including peptides,
antibodies, small organic molecules, carbohydrates, etc.
[0044] Agents specifically disclosed herein, as well as
derivatives, and peptidomimetics of said agents and agents
identified by design and/or screening assays find application in
increasing in the cell surface expression of degradation-prone CFTR
proteins and in the treatment of CF. Thus, methods for increasing
the cell surface expression of a degradation-prone CFTR and
treating cystic fibrosis are also provided by this invention.
[0045] In accordance with one embodiment, the cell surface
expression of a degradation-prone CFTR protein is enhanced or
increased by contacting a cell expressing a degradation-prone CFTR
with an agent that decreases or inhibits the interaction between
the CFTR protein and CAL so that the cell surface expression of the
CFTR protein is increased or enhanced. Desirably, the agent is
administered in an amount that effectively stabilizes the
degradation-prone CFTR protein and increases the amount of said
CFTR protein present or detectable at the cell surface by at least
60%, 70%, 80%, 90%, 95%, 99% or 100% as compared to cells not
contacted with the agent. Any cell can be employed in this method
of the invention so long as it expresses a degradation-prone CFTR.
Specific examples of such cells include, but are not limited to,
primary cells of a subject with CF or cultured airway epithelial
cell lines derived from a CF patient's bronchial epithelium (e.g.,
CFBE41O-). It is contemplated that this method of the invention can
be used to increase cell surface expression of a degradation-prone
CFTR protein in a human subject as well as increase the cell
surface expression of a degradation-prone CFTR protein in an
isolated cell or cell culture to, e.g., study the transport and/or
activity of the mutant protein at the cell surface.
[0046] In another embodiment, a subject with CF or at risk of CF is
treated with one or more the agents of the invention. In accordance
with this embodiment, an effective amount of an agent that
selectively inhibits the interaction between a degradation-prone
CFTR and CAL is administered to a subject in need of treatment
thereby preventing or treating the subject's cystic fibrosis.
Subjects benefiting from treatment with an agent of the invention
include subjects confirmed as having CF, subjects suspected of
having CF, or subjects at risk of having CF (e.g., subjects with a
family history).
[0047] Cystic Fibrosis is known to result from the dysfunction of
CFTR due to mutations in the gene. While the most common mutations
involve a deletion of phenylalanine in position 508, other
mutations have been described (Grasemann & Ratjen (2010) Expert
Opin. Emerg. Drugs. 15:653-659; Pettit & Johnson (2011) Ann.
Pharmacother. 45:49-59) These can be classified according to the
effect they have on the CFTR (Table 4). In one aspect, the subject,
benefiting from treatment in accordance with the present invention
expresses a degradation-prone CFTR (Class II mutation), such as
.DELTA.F508, .DELTA.I507 or N1303K.
TABLE-US-00004 TABLE 4 Class Description I Defective or absence of
CFTR protein synthesis with premature termination of CFTR
production II Impaired processing: typically a defect in protein
trafficking and degradation by the endoplasmic reticulum III
Defective regulation: the CFTR reaches the apical cell surface but
is not activated by ATP or cAMP IV Impaired function: transport of
chloride ions is reduced at the apical membrane V Reduced synthesis
of normal functioning CFTR Jones & Helm (2009) Drugs 69:
2003-2010; Grasemann & Ratjen (2010) supra; O'Sullivan &
Freedman (2009) Lancet 373: 1991-2004.
[0048] Successful clinical use of a selective inhibitor of the
invention can be determined by the skilled clinician based upon
routine clinical practice, e.g., by monitoring frequency of
respiratory infections and/or coughing; or changes in breathing,
abdominal pain, appetite, and/or growth according to methods known
in the art.
[0049] Agents disclosed herein can be employed as isolated and
purified molecules (i.e., purified peptides, derivatives, or
peptidomimetics), or in the case of peptides, be expressed from
nucleic acids encoding said peptides. Such nucleic acids can, if
desired, be naked or be in a carrier suitable for passing through a
cell membrane (e.g., DNA-liposome complex), contained in a vector
(e.g., plasmid, retroviral vector, lentiviral, adenoviral or
adeno-associated viral vectors and the like), symptoms of cystic
fibrosis. Other agents of use in the combination therapy include,
but are not limited to CFTR correctors, CFTR potentiators,
mucolytics and anti-inflammatory agents.
[0050] CFTR correctors are molecules that correct one or more
defects found in Class II mutations by rescuing proteins from
endoplasmic reticulum degradation, improving trafficking of CFTR to
the cell surface, and/or inhibiting proteins that are involved in
the recycling of CFTR in the cell membrane. Several correctors have
been identified using high throughput assays (O'Sullivan &
Freedman (2009) Lancet 373:1991-2004). For example, Ataluren
(3-[5-(2-Fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) can cause
ribosomal read-through of premature stop mutations in patients with
class I mutations, correct the processing of CFTR, and thereby
increase the production of functional CFTR (Jones & Helm (2009)
supra; Wilschanski, et al. (2011) Eur. Respir. J. 38:59-69).
Lumacaftor (VX-809;
3-{6-{[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropanecarbonyl-
]amino}-3-methylpyridin-2-yl}benzoic acid) is another corrector
that acts as a "chaperone" to assist the movement of defective CFTR
to the epithelial cell membrane (Jones & Helm (2009) supra;
O'Sullivan & Freedman (2009) supra). Indeed, it has been shown
that Lumacaftor can restore the P.sub.o of .DELTA.F508-CFTR to near
wild-type levels (Van Goor, et al. (2011) supra). Lumacaftor can be
provided in any suitable form including, but not limited to tablet,
capsule, injectable, or aerosol. Dosing of Lumacaftor can be in the
range of 200 to 600 mg once daily. Another corrector is corr-4a
(N-(2-(5-Chloro-2-methoxy-phenylamino)-4'-methyl-[4,5']bithiazolyl-2'-yl)-
-benzamide), which increases F508.DELTA.-CFTR cell-surface
expression and increases chloride conductance. As demonstrated
herein, iCAL36 peptide can enhance therapeutic efficacy of
correctors such as corr-4a.
[0051] A CFTR potentiator enhances the activity of CFTR that is
correctly located at the cell membrane. CFTR potentiators are
particularly useful in the treatment of subjects with class III
mutations. CFTR potentiators of use in this invention include
certain flavones and isoflavones, such as genistein, which are
capable of stimulating CFTR-mediated chloride transport in
epithelial tissues in a cyclic-AMP independent manner (See U.S.
Pat. No. 6,329,422, incorporated herein by reference in its
entirety); phenylglycine-01
(2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenyla-
cetamide); felodipine (Ethyl methyl
4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4-dihydro-3,5-pyridinedicarboxylate-
); sulfonamide SF-01
(6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid cycloheptylamide); and UCCF-152
(3-[2-(benzyloxy)phenyl]-5-(chloromethyl)isoxazole). Ivacaftor
(VX-770;
N-(2,4-Di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carbox-
amide) has also been shown to increase CFTR channel open
probability (P.sub.o) in both the F508.DELTA. processing mutation
and the G551D gating mutation (Van Goor, et al. (2011) supra).
Ivacaftor can be provided, e.g., in tablet form (KALYDECO; 150 mg
Ivacaftor) or alternatively in any other suitable form, e.g., as an
aerosol, capsule or injectable. Dosing of Ivacaftor can, e.g.,
include 250 mg Ivacaftor every 12 hours.
[0052] In some embodiments, the other agent is a single compound
with dual corrector and potentiator activities. Such agents include
VRT-532 (3-(2-hydroxy-5-methylphenyl)-5-phenylpyrazole) and
cyanoquinolines such as
N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-3-methoxybenzami-
de (CoPo-2), as well as hybrid bithiazole-phenylglycine
corrector-potentiators which, when cleaved by intestinal enzymes,
yield an active bithiazole corrector and phenylglycine potentiator
(Mills, et al. (2010) Bioorg. Med. Chem. Lett. 20:87-91).
[0053] Mucolytics are agents that dissolve thick mucus by
dissolving various chemical bonds within secretions, which in turn
can lower the viscosity by altering the mucin-containing
components. Mucolytics of use in this invention include, but are
not limited to acetylcysteine ((2R)-2-acetamido-3-sulfanylpropanoic
acid), ambroxol
(trans-4-(2-Amino-3,5-dibrombenzylamino)-cyclohexanol), bromhexine
(2,4-dibromo-6-{[cyclohexyl(methyl)amino]methyl}aniline),
carbocisteine (R)-2-Amino-3-(carboxymethylsulfanyl)propanoic acid),
domiodol ([2-(iodomethyl)-1,3-dioxolan-4-yl]methanol), dornase alfa
(recombinant human deoxyribonuclease I), eprazinone
(3-[4-(2-ethoxy-2-phenyl-ethyl)piperazin-1-yl]-2-methyl-1-phenyl-propan-1-
-one), erdosteine
(2-[(2-oxothiolan-3-yl)carbamoylmethylsulfanyl]acetic acid),
letosteine
(2-{2-[(2-ethoxy-2-oxoethyl)thio]ethyl}-1,3-thiazolidine-4-carboxylic
acid), mannitol, mesna (sodium 2-sulfanylethanesulfonate),
neltenexine
(N-(2,4-dibromo-6-{[(4-hydroxycyclohexyl)amino]methyl}phenyl)thiophene-2--
carboxamide), and sobrerol
((1S)-5-(1-hydroxy-1-methylethyl)-2-methylcyclohex-2-en-1-ol),
stepronin (N-{2-[(2-thienylcarbonyl)thio]propanoyl}glycine).
[0054] Inflammation is a major component of cystic fibrosis. If
untreated, inflammation can irreversibly. damage the airways,
leading to bronchiectasis and ultimately respiratory failure.
Anti-inflammatory drugs used in the treatment of cystic fibrosis
include steroids such as corticosteroids and nonsteroidal
anti-inflammatory drugs such as ibuprofen. Other agents include
pentoxifylline and azithromycin, which, in addition to its
antimicrobial effects, also possesses anti-inflammatory
properties.
[0055] Other therapeutics of use in combination with the agents of
this invention include, but are not limited to, 2,2-dimethyl
butyric acid (U.S. Pat. No. 7,265,153); glycerol, acetic acid,
butyric acid, D- or L-amino-n-butyric acid, alpha- or
beta-amino-n-butyric acid, arginine butyrate or isobutyramide, all
disclosed in U.S. Pat. Nos. 4,822,821 and 5,025,029; and butyrin,
4-phenyl butyrate, phenylacetate, and phenoxy acetic acid,
disclosed in U.S. Pat. No. 4,704,402.
[0056] The combination therapy of this invention preferably
includes (a) at least one agent that selectively inhibits the
interaction between a degradation-prone CFTR and CAL and (b) a CFTR
corrector, CFTR potentiator, mucolytic, anti-inflammatory agent, or
combination thereof. In some embodiments, the combination therapy
of this invention includes (a) at least one agent that selectively
inhibits the interaction between a degradation-prone CFTR and CAL
and (b) a CFTR corrector, CFTR potentiator, or combination thereof.
In accordance with this invention, the active agents of the
combination therapy can be administered simultaneously of
consecutively, within seconds, minutes, hours, days or weeks of
each other. It is expected that the above-referenced combination
therapy will have an additive or synergistic effect in the
treatment of cystic fibrosis. In particular, it is expected that
the combination of a selective inhibitor of the CFTR and CAL
interaction, a CFTR corrector, and a CFTR potentiator will reverse
all three defects (folding, gating, and stability) of
.DELTA.F508-CFTR.
[0057] The present invention also provides a kit containing (a) an
agent for inhibiting the interaction between a degradation-prone
CFTR and CAL in combination with (b) a CFTR corrector, CFTR
potentiator, mucolytic, anti-inflammatory agent, or combination
thereof, for use in the prevention or treatment of cystic fibrosis.
In some embodiments, the kit includes a plurality of separate
containers, each containing at least one active agent useful in a
combination therapy for the prevention or treatment of cystic
fibrosis. The kit contains a first container containing an agent
for inhibiting the interaction between a degradation-prone CFTR and
CAL. The kit further includes a container for a CFTR corrector, a
container for a CFTR potentiator, a container for a mucolytic, and
or a container for an anti-inflammatory agent. The containers of
the kit may be enclosed within a common outer packaging, such as,
for example a cardboard or plastic box or a shrink wrap outer skin
enclosing the various containers. In certain embodiments, the agent
for inhibiting the interaction between a degradation-prone CFTR and
CAL; and CFTR corrector, CFTR potentiator, mucolytic, and/or
anti-inflammatory agent are each individually formulated in an
acceptable carrier. The kit may be in the form of a consumer
package or prescription package which provides the products
described above. The package may provide instructions or directions
on how to use and/or combine the products for one or more treatment
regimens.
[0058] For therapeutic use, active agents of the invention can be
formulated with a pharmaceutically acceptable carrier at an
appropriate dose. Such pharmaceutical compositions can be prepared
by methods and contain carriers which are well-known in the art. A
generally recognized compendium of such methods and ingredients is
Remington: The Science and Practice of Pharmacy, Alfonso R.
Gennaro, editor, 20th ed. Lippincott Williams & Wilkins:
Philadelphia, Pa., 2000. A pharmaceutically acceptable carrier,
composition or vehicle, such as a liquid or solid filler, diluent,
excipient, or solvent encapsulating material, is involved in
carrying or transporting the subject agent from one organ, or
portion of the body, to another organ, or portion of the body. Each
carrier must be acceptable in the sense of being compatible with
the other ingredients of the formulation and not injurious to the
patient.
[0059] Examples of materials which can serve as pharmaceutically
acceptable carriers include sugars, such as lactose, glucose and
sucrose; starches, such as corn starch and potato starch;
cellulose, and its derivatives, such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered
tragacanth; malt; gelatin; talc; excipients, such as cocoa butter
and suppository waxes; oils, such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; pH buffered
solutions; polyesters, polycarbonates and/or polyanhydrides; and
other non-toxic compatible substances employed in pharmaceutical
formulations. Wetting agents, emulsifiers and lubricants, such as
sodium lauryl sulfate and magnesium stearate, as well as coloring
agents, release agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants can also be
present in the compositions.
[0060] Compositions of the present invention can be administered
parenterally (for example, by intravenous, intraperitoneal,
subcutaneous or intramuscular injection), topically including via
inhalation, transdermally, orally, intranasally, intravaginally, or
rectally according to standard medical practices.
[0061] The selected dosage level of an agent will depend upon a
variety of factors including the activity of the particular agent
of the present invention employed, the route of administration, the
time of administration, the rate of excretion or metabolism of the
particular agent being employed, the duration of the treatment,
other drugs, compounds and/or materials used in combination with
the particular agent employed, the age, sex, weight, condition,
general health and prior medical history of the patient being
treated, and other factors well-known in the medical arts.
[0062] A physician having ordinary skill in the art can readily
determine and prescribe the effective amount of the pharmaceutical
composition required based upon the administration of similar
compounds or experimental determination. For example, the physician
could start doses of an agent at levels lower than that required in
order to achieve the desired therapeutic effect and gradually
increase the dosage until the desired effect is achieved. This is
considered to be within the skill of the artisan and one can review
the existing literature on a specific agent or similar agents to
determine optimal dosing.
[0063] The fact that other proteins destined for the intracellular
transport pathway frequently exhibit transport delays due to
mutations, or other factors, indicates that the cell-surface
expression of such degradation-prone proteins may also be mediated
by CAL. Thus, it is contemplated that the agents of this invention
can also be used to induce or increase the cell surface expression
of other degradation-prone proteins. Accordingly, physiological
disorders associated with other degradation-prone proteins besides
CFTR can similarly be treated using the methods disclosed herein.
Physiological disorders associated with a degradation-prone protein
that can be treated in a method of the invention include, for
example, Stargardt's disease and particular types of macular
dystrophy caused by mutations of the retinal rod transporter,
ABC-R, resulting in deficiency of export.
[0064] The invention is described in greater detail by the
following non-limiting examples.
Example 1
Materials and Methods
[0065] Protein Expression and Purification.
[0066] CALP (UniProt Accession No. Q9HD26-2) was expressed and
purified (Cushing, et al. (2008) Biochemistry 47:10084-10098).
TIP-1 (Accession No. 014907) was expressed and purified similarly
except that an N-terminal His.sub.10 tag was used with a HRV 3C
protease recognition sequence (LEVLFQ*G; SEQ ID NO:35) upstream of
the full-length protein sequence. Following TIP-1 purification via
immobilized metal-affinity chromatography, the protein was injected
onto a SUPERDEX S75 gel filtration column (GE Healthcare)
equilibrated in 50 mM Tris pH 8.5, 150 mM NaCl, 0.1 mM TCEP, 0.02%
NaN.sub.3. Human rhinovirus 3C protease (Novagen) was added to the
protein at a 1:30 mass ratio and incubated at 4.degree. C. for 48
hours. Following cleavage, the protein was passed through a 1 mL
HISTRAP HP column (GE Healthcare) equilibrated in 20 mM imidazole,
25 mM Tris pH 8.5, 150 mM NaCl, 0.1 mM TCEP, 0.02% NaN.sub.3. The
protein was further purified on a SUPERDEX S75 column as described
above. Following gel filtration, the protein was dialyzed into gel
filtration buffer with 5% glycerol. TIP-1 protein quantitation was
achieved by using the A.sub.280 nm experimentally determined
extinction coefficient value of 10715 cm.sup.-1*M.sup.-1 (Cushing,
et al. (2008) supra). All purified proteins were deemed thermally
stable at the temperatures used for in vitro binding measurements
by monitoring thermal stability (Cushing, et al. (2008) supra).
[0067] Peptide Synthesis.
[0068] All peptides, except those used in peptide arrays
experiments, were synthesized and HPLC-purified by Tufts Peptide
Core Facility. Peptides with N-terminally coupled fluorescein (via
an aminohexanoic acid linker) are denoted by a "F*-" prefix.
Biotin-conjugated peptides ("BT") were N-terminally coupled via an
WrFKK (SEQ ID NO:34) linker sequence (r=D-Arg).
[0069] Cell Culture.
[0070] CFBE410-cells (Bruscia, et al. (2002) Gene Ther 9:683-685)
stably expressing .DELTA.F508-CFTR under the control of a
cytomegalovirus promoter (CFBE-.DELTA.F cells; Li, et al. (2006)
Am. J. Respir. Cell Mol. Biol. 34:600-608) are described in the art
(Bebok, et al. (2005) J. Physiol. 569:601-615). Cells were cultured
and switched to MEM containing only penicillin and streptomycin 24
hours before experiments. All cells used in experiments were
between passages 15 and 20.
[0071] PDZ Pull-Down Assay.
[0072] Briefly, pull-down assays were performed by incubating
biotin-conjugated peptides or buffer with streptavidin paramagnetic
beads (PROMEGA). Excess peptide was removed by washing. Clarified
CFBE-.DELTA.F cell lysates were added to the beads and incubated
with rotation for 90 minutes at 4.degree. C. Beads were washed and
bound proteins were eluted with buffer, peptide inhibitor, or
scrambled peptide. Proteins were separated by SDS-PAGE and
immunoblotted. For mass spectrometry identification, SILVERQUEST
(Invitrogen) was used to silver stain protein bands according to
manufacturer's instructions. Bands were considered to be candidate
protein interactors if they were enriched in the specific
non-biotinylated peptide-eluted lane (e.g. iCAL36) versus the
SCR-eluted lane. Destained protein bands were identified.
Confirmation of peptide fragments was from three independent sample
submissions, with CAL and TIP-1 positively identified with each
submission.
[0073] Fluorescence Anisotropy and Peptide Array Experiments.
[0074] Peptide fluorescence anisotropy binding studies were
performed as described (Cushing, et al. (2008) supra). LAG values
were calculated from K.sub.i values. In the case of weak affinity
inhibitors (K.sub.i>1000 .mu.M), K.sub.i values were estimated.
Inverted peptide array experiments were performed as described
(Boisguerin, et al. (2004) Chem. Biol. 11:449-459; Boisguerin, et
al. (2007) Chembiochem. 8:2302-2307). For TIP-1 peptide array
experiments, His.sub.10 tagged (uncleaved) protein was used to
facilitate quantitation.
[0075] TIP-1:iCAL36 Crystallization and Data Collection.
[0076] iCAL36 was added at a final concentration of 1 mM to
purified TIP-1 at 5.5 mg ml.sup.-1 in 10 mM HEPES pH 7.4, 25 mM
NaCl. Initial crystallization conditions were identified for the
TIP-1:iCAL36 complex by micro-batch screening at the
Hauptman-Woodward Medical Research Institute High-Throughput
Screening laboratory (Luft, et al. (2003) J. Struct. Biol.
142:170-179). Crystallization conditions identified by screening
were optimized in hanging-drop format at 291 K, by adding 2 .mu.l
of the complex in screening buffer (5.5 mg ml.sup.-1 TIP-1 and 1 mM
iCAL36) to 2 .mu.l reservoir solution. The reservoir contained 500
.mu.l solution. Crystals appeared in 2-4 days and continued to grow
for up to 14 days. The crystal used for data collection was
obtained using 100 mM NH.sub.4SCN, 100 mM MES pH 6.0, 36% (w/v)
polyethylene glycol (PEG) 1000 as reservoir buffer.
[0077] For data collection, the crystal was transferred into
cryoprotectant buffer [200 mM N.sub.H4SCN, 100 mM MES pH 6.0, 30%
(w/v) PEG 400. The data set used for structure determination was
obtained at 100K, .lamda.=1.0000 .ANG. on beam line X6A at the
National Synchrotron Light Source (NSLS) at Brookhaven National
Laboratory. Two data sets were collected and later merged; one over
a 360.degree. range, using 0.3.degree. frames and an exposure time
of 2 seconds per frame, and the second over 60.degree., using
1.degree. frames, an exposure time of 0.5 seconds per frame, and an
aluminum foil filter. Diffraction data were processed using the XDS
package. The TIP-1:iCAL36 complex crystallizes in space-group P1
with unit cell dimensions a=27.0, b=34.1, c=66.9 .ANG.,
.alpha.=79.6.degree., .beta.=87.1.degree., .gamma.=89.9.degree.,
and diffracted to a resolution of 1.24 .ANG..
[0078] TIP-1:iCAL36 Structure Determination and Refinement.
[0079] Molecular replacement was performed using PHENIX (Adams, et
al. (2010) Acta Crystallogr. D Biol. Crystallogr. 66:213-221;
McCoy, et al. (2007) J. Appl. Crystallogr. 40:658-674), using the
TIP-1:.beta.-catenin structure as a template (PDB ID=3DIW; Zhang,
et al. (2008) J. Mol. Biol. 384:255-263). The model was built and
refined using phenix (Adams, et al. (2010) supra). The final
structure has a R.sub.work=18.0% and R.sub.free=19.3%. Detailed
data collection and refinement statistics are in Table 5.
TABLE-US-00005 TABLE 5 Data Collection Space Group P1 Unit cell
dimensions: a, b, c (.ANG.) 26.97, 34.09, 66.86 .alpha., .beta.,
.gamma. (.degree.) 79.64, 87.15, 89.97 Matthews Coefficient
(.ANG..sup.3 Da.sup.-1) 2.01 Molecules in ASU (Z) 2 Solvent content
0.39 Wavelength (.ANG.) 0.9181 Resolution.sup.a (.ANG.) 19.11-1.24
(1.31-1.24) Unique reflections 60547 R.sub.sym.sup.b 0.04 (0.27)
<I/.sigma..sub.I> 27.63 (4.29) R.sub.mrgd-F.sup.c 5.5 (43.9)
Completeness (%) 91.3 (70.0) Molecular Replacement Rotation
function search Peak no. 0 Log-likelihood gain 191 Z-score 15.4
Translation function search Peak no. 2 Log-likelihood gain 749
Z-score 22.9 Overall log-likelihood gain 1476 Refinement Total
number of reflections 60,539 Reflections in the test set 3,044
R.sub.work.sup.d/R.sub.free.sup.e 0.180/0.193 Number of atoms:
Protein 1,923 Solvent 256 Ramachandran plot.sup.f (%) 92.1/7.9/0/0
B.sub.av (.ANG..sup.2) Protein 19.75 Solvent 28.01 Bond length RMSD
0.005 Bond angle RMSD 0.985 .sup.aValues in parentheses are for
data in the highest-resolution shell. .sup.bRsym = .SIGMA.hi |I(h)
- Ii(h)|/hi Ii(h), where Ii(h) and I(h) values are the i-th and
mean measurements of the intensity of reflection h, respectively.
.sup.cSigAno = <(|F(+) - F(-)|/.sigma..sub..DELTA.)>.
.sup.dRwork = .SIGMA.h |Fobs(h) - Fcalc(h)|/.SIGMA.h Fobs(h),
h.epsilon. {working set}. .sup.eRfree = h _Fobs(h) _ Fcalc(h)_/h
Fobs(h), h.epsilon. {test set}. .sup.fCore/allowed/generously
allowed/disallowed.
[0080] TIP-1:iCAL42 and TIP-1:.beta.-Catenin Substitution
Modeling.
[0081] The TIP-1:iCAL36 and TIP-1:.beta.-catenin structures were
aligned using PyMOL (RMSD=0.38 .ANG.).
[0082] TIP-1:.beta.-Catenin Substitution Modeling.
[0083] The TIP-1:.beta.-catenin cocrystal structure (PDB ID=3DIW;
Zhang, et al. (2008) supra) was used as a template for assessing
the observed loss of affinity in the iCAL42 Trp-Leu P.sup.-5 ligand
substitution. The P.sup.-5 tryptophan was substituted for leucine
in WINCOOT (Emsley, et al. (2010) Acta Crystallogr. D Biol.
Crystallogr. 66:486-501), and individual PDB files created for the
possible rotomers. Each rotamer was evaluated for potential steric
clashes and non-optimal bond geometry with MOLPROBITY (Chen, et al.
(2010) Acta Crystallogr. D Biol. Crystallo Ussing Chamber gr.
66:12-21).
[0084] Measurements.
[0085] Short circuit current (I.sub.SC) measurements were
performed. Briefly, 10.sup.5 cells were seeded onto 12 mm SNAPWELL
permeable supports (Corning) and allowed to form polarized
monolayers over the course of 9 days. CFBE-.DELTA.F cells were
dosed with 0.5 mM peptide via BIOPORTER (Sigma) 3.5 hour before the
start of Ussing chamber measurements. Cells were maintained at
37.degree. C. throughout treatments; the DMSO concentration did not
exceed 0.03%. Cells were treated sequentially with 50 .mu.M
amiloride, 20 .mu.M forskolin, 50 .mu.M genistein, and 5 .mu.M
CFTR.sub.inh-172 in 5.0 minute intervals. CFTR-specific chloride
efflux was computed as the magnitude of .DELTA.I.sub.SC following
application of CFTR.sub.inh-172. Resistances were monitored
throughout each experiment to ensure monolayer integrity.
[0086] Statistical Analysis.
[0087] Values are reported as mean.+-.SD except for Ussing chamber
experiments where mean.+-.SEM is reported. Student's one-tailed
t-test was used for fluorescence anisotropy binding experiments
while the Student's one-tailed paired t-test was used for analysis
of Ussing chamber experiments.
[0088] Computational Designs with K*.
[0089] The previously-determined NMR structure of the CAL PDZ
domain bound to the C-terminus of CFTR was used to model the
binding of CAL to CFTR (Piserchio, et al. (2005) Biochemistry
44:16158-16166). The CFTR peptide in the NMR structure was
truncated to the six most C-terminal amino acids and mutated to the
amino acid sequence WQTSII (SEQ ID NO:36) to mimic the best peptide
hexamer for CAL discovered thus far. An acetyl group was modeled
onto the N-terminus of the peptide using restrained molecular
dynamics and minimization where the N-terminus of the peptide was
allowed to move, while the remainder of the protein complex was
restrained using a harmonic potential (Case, et al. (2005) J. Comp.
Chem. 26:1668-1688). An 8 .ANG. shell around the peptide hexamer
was used as the input structure to K*. The four most C-terminal
residues, TSII (SEQ ID NO:37), were allowed to mutate to the
following residues during the design search: Thr (all amino acids
except Pro), Ser (T/S), Ile (all amino acids except Pro), and Ile
(I/L/V). In addition, the Probe program (Word, et al. (1999) J.
Mol. Biol. 285:1711-1733) was used to determine the side-chains on
CAL that interact with the CFTR peptide mimic. The nine residues
that interact with the peptide, as well as the two most N-terminal
residues on the peptide, were allowed to be flexible during the
design search. The peptide was allowed to rotate and translate as a
rigid body during the search, as previously described for small
molecules (Chen, et al. (2009) supra; Georgiev, et al. (2008)
supra; Frey, et al. (2010) Proc. Natl. Acad. Sci. USA
107:13707-13712). To explore the feasibility of the new algorithms,
unless otherwise noted, full partition functions were not computed
and a maximum of 10.sup.3 conformations were allowed to contribute
to each partition function.
[0090] Peptide Array Comparison.
[0091] The peptide array data was composed of 6223 C-termini
(11-mers) from human proteins. The array was incubated with the CAL
PDZ domain in order to determine binding of CAL to the 11-mers. The
K* algorithm was used to evaluate 4-mer structural models of the
peptide-array sequences to verify the accuracy of the
predictions.
[0092] To compare the array data with the K* predictions, the
quantitative array data, measured in biochemical light units
(BLUs), was converted into a binary yes/no CAL binding event. In
other words, by setting a binding cutoff on the peptide array, each
sequence was classified as either a CAL binder or non-binder. The
cutoff value was chosen as three standard deviations away from the
average BLU value of the array.
[0093] Prospective Computational Predictions.
[0094] K* was used to search over all peptide sequences within the
CAL PDZ domain sequence motif to find new CAL peptide inhibitors.
For computational efficiency, the number of conformations
enumerated by A* for each partition function was limited to
10.sup.3 conformations. Two sets of peptides (promising designs and
poorly ranked designs) were chosen to be experimentally
validated.
[0095] In order to choose the most promising peptide inhibitors, a
second K* design was performed, where K* scores for the top 30
sequences were re-calculated with the number of enumerated
conformations per partition function increased to 10.sup.5. Several
top-ranked sequences were chosen to be experimentally tested.
First, the top seven ranked sequences from the second run were
chosen. In addition, two sequences that greatly increased in
ranking from the first to second run (rank 29 to 9, and rank 28 to
11) were chosen as well. Finally, a K* run was conducted using
Charmm forcefield parameters instead of Amber parameters. Two
sequences that scored high on both the Amber and Charmm runs were
chosen to be experimentally tested as well.
[0096] The poorly-ranked designs were chosen to minimize the
sequence similarity among the set of poorly-ranked peptides. First,
the worst-ranked peptide was chosen and added to initialize the set
of negative sequences. Next, sequences were successively chosen
from the worst 200 K* ranked sequences and added to the set in
order to maximize the amino acid sequence diversity with all the
sequences already in the set. The similarity between two sequences
was determined using the PAM-30 similarity matrix (Dayhoff, et al.
(1978) Nat. Biomed. Res. Found. 5:345-352). In total, 23 (eleven
top-ranked and twelve poorly-ranked) K*-computed peptide inhibitor
sequences were experimentally tested.
[0097] Experimental Procedure.
[0098] The experimental inhibitory constants of top- and
poorly-ranked peptide sequences from the K* CAL-CFTR design were
experimentally determined. As a control, the best known peptide
hexamer was also retested. The corresponding N-terminally
acetylated peptides were purchased from NEO Bio-Science (Cambridge,
Mass.) and the K.sub.i values for the peptides were detected using
fluorescence polarization. Briefly, the CAL PDZ domain was
incubated with a labeled peptide of known binding affinity. Each
peptide inhibitor was serially diluted and the protein-peptide
mixture was added to each dilution. Finally, the amount of
competitive inhibition was tracked using residual fluorescence
polarization.
[0099] The Ussing chamber experiments were performed as described
herein. Polarized monolayers of patient-derived bronchial
epithelial cells, CFBE-.DELTA. cells, were treated with peptide and
BIOPORTER (Gene Therapy Systems; San Diego, Calif.) delivery agent.
Peptide inhibitor was applied to the monolayer and the short
circuit currents (I.sub.SC) were monitored in Ussing chambers.
.DELTA.F508-CFTR chloride flux was measured as the change in
I.sub.sc when the CFTR specific inhibitor, CFTR.sub.inh-172
(Taddel, et al. (2004) FEBS let. 558:52-56; Ma, et al. (2002) J.
Clin. Invest. 110:1651-1658), was applied to the cell
monolayer.
Example 2
Identification of Selective Inhibitors of the CAL and CFTR
Interaction
[0100] Using peptide-array screening and fluorescence-polarization
binding assays, a series of peptide sequences were identified that
bind CAL progressively more tightly than CAL binds to CFTR, and
that in parallel bind NHERF1 and NHERF2 progressively more weakly
than these proteins bind to CFTR.
[0101] To test the ability of CAL inhibitors to rescue CFTR,
cultured airway epithelial cells (cell line CFBE41o-, derived from
a CF patient's Bronchial Epithelium) were grown on filters,
permitting formation of polarized cell monolayers similar to those
found in epithelial tissues. The CFBE41o-cell line is
well-recognized as an airway epithelial model system for the study
of CF processes. These cells express the most common disease mutant
associated with CF, .DELTA.F508-CFTR, which is characterized by the
loss of a single amino acid codon at position 508 of CFTR. Roughly
50% of CF patients are homozygous for .DELTA.F508-CFTR, and another
40% are heterozygotes for this allele. Functional rescue of
.DELTA.F508-CFTR therefore has the potential to alleviate symptoms
in up to 90% of CF patients. Although very little .DELTA.F508-CFTR
protein is synthesized in the absence of intervention, the protein
itself retains some functional activity. If rescued and stabilized
it can restore physiological CFTR activity, potentially reversing
the processes that lead to chronic lung infection, and ultimately
death, in most CF patients.
[0102] When introduced into CFBE41o-cells using commercial peptide
transfection reagents, representative peptide and peptidomimetic
compounds were able to increase the amount of .DELTA.F508-CFTR
protein at the apical membrane and to increase the CFTR-mediated
chloride efflux across the monolayers. The magnitude of the
functional rescue correlated with the selectivity of the peptides
for CAL vs. NHERF1 and NHERF2; the more selective the peptide for
the CAL binding site, the more effective it was at enhancing
chloride efflux.
[0103] Furthermore, when used in combination with a compound that
enhances the biosynthesis of .DELTA.F508-CFTR (a "corrector"), the
instant inhibitors showed an additive effect, comparable in
magnitude to that of the corrector compound.
[0104] Although compounds have previously been designed to enhance
the synthesis and/or chloride-channel activity of CFTR, the instant
inhibitors were designed to stabilize mutant CFTR protein that has
already been synthesized within the cell and successfully
transported to the cell surface. The peptides and peptidomimetics
disclosed herein provide a basis for further optimization of CAL
inhibitor properties in terms of affinity and selectivity for CAL,
in vivo proteolytic stability, cellular uptake, and ADME
characteristics.
Example 3
Assays for Assessing Activity of Selective Inhibitors
[0105] Agents of the present invention can be assayed for their
ability to stimulate chloride transport in epithelial tissues. Such
transport may result in secretion or absorption of chloride ions.
The ability to stimulate chloride transport may be assessed using
any of a variety of systems. For example, in vitro assays using a
mammalian trachea or a cell line, such as the permanent airway cell
line Calu-3 (ATCC Accession Number HTB55) may be employed.
Alternatively, the ability to stimulate chloride transport may be
evaluated within an in vivo assay employing a mammalian nasal
epithelium. In general, the ability to stimulate chloride transport
may be assessed by evaluating CFTR-mediated currents across a
membrane by employing standard Ussing chamber (see Ussing &
Zehrahn (1951) Acta. Physiol. Scand. 23:110-127) or nasal potential
difference measurements (see Knowles, et al. (1995) Hum. Gene
Therapy 6:445-455). Within such assays, an agent that stimulates a
statistically significant increase in chloride transport at a
concentration of about 1-300 .mu.M is said to stimulate chloride
transport.
[0106] Within one in vitro assay, the level of chloride transport
may be evaluated using mammalian pulmonary cell lines, such as
Calu-3 cells, or primary bovine tracheal cultures. In general, such
assays employ cell monolayers, which may be prepared by standard
cell culture techniques. Within such systems, CFTR-mediated
chloride current may be monitored in an Ussing chamber using intact
epithelia. Alternatively, chloride transport may be evaluated using
epithelial tissue in which the basolateral membrane is
permeabilized with Staphylococcus aureus .alpha.-toxin, and in
which a chloride gradient is imposed across the apical membrane
(see Illek, et al. (1996) Am. J. Physiol. 270:C265-75). In either
system, chloride transport is evaluated in the presence and absence
of a test agent, and those compounds that stimulate chloride may be
used within the methods provided herein.
[0107] Within another in vitro assay for evaluating chloride
transport, cells, such as NIH 3T3 fibroblasts, are transfected with
a CFTR gene having a mutation associated with cystic fibrosis
(e.g., .DELTA.F508-CFTR) using well known techniques (see Anderson,
et al. (1991) Science 25:679-682). The effect of an agent on
chloride transport in such cells is then evaluated by monitoring
CFTR-mediated currents using the patch clamp method (see Hamill, et
al. (1981) Pflugers Arch. 391:85-100) with and without agent.
[0108] Alternatively, such assays may be performed using a
mammalian trachea, such as a primary cow tracheal epithelium using
the Ussing chamber technique as described above. Such assays are
performed in the presence and absence of a test agent to identify
agents that stimulate chloride transport.
Example 4
Single-Domain Specificity of a CAL PDZ Inhibitor that Rescues
.DELTA.F508-CFTR
[0109] iCAL36 is a Highly Selective PDZ Inhibitor.
[0110] To determine the full spectrum of PDZ domains inhibited by
iCAL36 (sequence: ANSRWPTSII; SEQ ID NO:20) in epithelial cells, a
pull-down/mass-spectrometry assay for iCAL36 interactors was
developed. As bait, an N-terminally biotinylated (BT-) version of
iCAL36 was used, which retained the binding profile of the decamer.
BT-iCAL36 was coupled to streptavidin beads and incubated with
whole-cell lysates (WCL) from human cystic fibrosis bronchial
epithelial cells expressing .DELTA.F508-CFTR (CFBE-.DELTA.F cells).
Mass spectrometry revealed only two PDZ proteins among the "prey"
proteins that were enriched in iCAL36 vs. control eluates. CAL was
identified with good peptide coverage. The second PDZ sequence
identified by mass-spectrometry was the Tax-interacting protein-1
(TIP-1). Both interactions were validated using WCL pull-downs and
immunoblot analysis. Thus, although initially engineered to avoid
interactions only with the NHERF1 and NHERF2 PDZ domains, iCAL36
has a strikingly selective interaction profile, robustly engaging
only a single "off-target" protein among the entire spectrum of PDZ
proteins present in airway epithelial cell lysates.
[0111] The significant enrichment of the iCAL36-eluted bands over
the inputs, especially in the case of TIP-1, was consistent with a
potent interaction. To quantify its strength relative to the
on-target binding of CAL, recombinant expression and purification
protocols were developed for the TIP-1 PDZ domain and its
interaction with a fluoresceinated iCAL36 peptide (F*-iCAL36) was
monitored by means of fluorescence polarization (FP). Titration
revealed a strong, dose- and sequence-dependent binding isotherm,
with a fitted K.sub.d of 0.54 .mu.M. Surprisingly, TIP-1 actually
bound F*-iCAL36 2.5-fold more tightly than CAL (K.sub.d=1.3 .mu.M),
and its submicromolar interaction placed it at the high-affinity
end of the spectrum of PDZ:peptide interactions (Stiffler et al.
(2007) Science 317:364-369).
[0112] An unusual protein composed almost entirely of a single PDZ
domain, TIP-1 has been implicated in negatively regulating the Wnt
signaling pathway by sequestering .beta.-catenin (Kanamori, et al.
(2003) J. Biol. Chem. 278:38758-38764). Recent reports also suggest
TIP-1 may play a role in regulating the surface expression of
membrane proteins, including Kir 2.3 (Alewine, et al. (2006) Mol.
Biol. Cell 17:4200-4211). Thus, despite the excellent overall
specificity of iCAL36, its off-target interaction with TIP-1 could
potentially have contributed to its effects on CFTR stability. To
resolve this target ambiguity, and to test the ability to achieve
true single-PDZ specificity, CAL inhibitors were designed without
TIP-1 affinity.
[0113] Sequence Determinants of the iCAL36:TIP-1 Interaction.
[0114] As a basis for eliminating the off-target interaction,
parallel structural and biochemical approaches were undertaken to
understand the contributions of individual iCAL36 side chains to
TIP-1 binding. To visualize the stereochemistry of binding, the
structure of the TIP-1:iCAL36 complex was determined by X-ray
crystallography. The iCAL36 peptide adopted a canonical PDZ-binding
conformation in the TIP-1 binding pocket, with standard C-terminal
carboxylate, P.sup.0 and P.sup.-2 interactions. In addition, the
P.sup.-5 side chain was bound within a deep, hydrophobic pocket
that provided excellent stereochemical complementarity to the
planar Trp-conjugated ring system. In contrast, the structure of
the CAL PDZ domain showed no equivalent pocket.
[0115] In order to assess the free-energy contribution of each side
chain to the interaction, substitutional analysis (SubAna) was
performed by synthesizing peptide arrays containing the iCAL36
sequence with the amino acid at each position individually replaced
with all 19 natural alternatives. Consistent with the
stereochemistry of the interaction, the binding patterns of the CAL
and TIP-1 PDZ domains also highlighted the importance of the
P.sup.-5 Trp side chain to the off-target binding affinity of
iCAL36. P.sup.-5 substitution with any other natural amino acid
abrogated TIP-1 binding, whereas multiple substitutions were
tolerated at other positions along the iCAL36 sequence. In
contrast, CAL binding was retained for multiple substitutions at
both the P.sup.-5 position and elsewhere in the sequence. Both the
biochemical and structural data thus indicated that the affinity of
TIP-1 for iCAL36 was tightly focused on the P.sup.-5 position,
whereas CAL's affinity was more broadly distributed along the
length of the peptide.
[0116] To identify the sources of iCAL36 affinity for TIP-1 in more
detail, the TIP-1 binding affinity of the somatostatin receptor
subtype 5 (SSR5) C-terminal peptide (ANGLMQTSKL; SEQ ID NO:38) was
also determined, which was the starting sequence for the original
peptide engineering effort. Using F*-iCAL36 as a high-affinity
reporter peptide, an FP displacement assay revealed that the SSR5
sequence interacted with TIP-1 even though it had a Met at the
P.sup.-5 position, a substitution that abrogated TIP-1 binding in
the context of the iCAL36 sequence. In comparison to unlabeled
iCAL36, which binds TIP-1 with a K.sub.i of 1.8 .mu.M, the K.sub.i
for the unlabeled SSR5 peptide binding was 130 .mu.M. Taken
together, these data indicate that both the baseline affinity of
the SSR5 starting sequence and the P.sup.-5 Trp represented
potential contributors to the high affinity of the off-target
interaction.
[0117] A Stereochemical Achilles' Heel.
[0118] The ability of the combinatorial peptide-array/FP
counterscreening paradigm to improve the iCAL selectivity profile
was analyzed. CombLib peptide arrays, in which all 400 possible
pairs of amino acids were inserted into positions P.sup.-5 and
P.sup.-4 had already been evaluated for binding to the CAL and
NHERF PDZ domains as described herein. A comparable CombLib was
subsequently prepared and surveyed for TIP-1 binding. In the
framework of the iCAL36 sequence, TIP-1 binding was strictly
confined to peptides that included an aromatic residue at P.sup.-5.
Parallel CombLibs based on the full iCAL36 sequence confirmed that
the P.sup.-5 and P.sup.-4 preferences were relatively independent
of upstream sequence context.
[0119] Comparison with published arrays identified a number of
combinations that bound CAL, but did not bind TIP-1 or any of the
NHERF domains studied. Among these was a Leu/Pro combination. The
SubAna arrays showed that the CAL-binding signal of the P.sup.-5
Leu substitution was comparable to those of the strongest Trp/Xaa
combinations. Separate SubAna arrays based on the new sequence
(iCAL42; ANSRLPTSII; SEQ ID NO:21) confirmed that the CAL PDZ
binding preferences were largely retained. Underscoring the
critical contribution of the P.sup.-5 Trp side chain, TIP-1 binding
was abrogated for all single substitutions of the Leu-based iCAL42
sequence except for the Leu/Trp revertant.
[0120] In order to quantitate the impact of the P.sup.-5 Leu
substitution and to assess inhibitory potential at high peptide
concentrations, FP displacement assays were performed. Consistent
with the qualitative data, CAL displacement isotherms showed that
iCAL42 retained robust CAL PDZ affinity, with a fitted K.sub.i
value of 53 .mu.M, only three-fold weaker than unlabeled iCAL36.
The NHERF CombLib preferences were also validated: iCAL42 failed to
bind any of the four NHERF1 or NHERF2 PDZ domains with appreciable
affinity. Critically, the iCAL42 displacement isotherm for TIP-1
was also essentially indistinguishable from the vehicle control up
to millimolar peptide concentrations, representing a >1500-fold
decrease in binding affinity. Thus, in the context of the iCAL36
sequence, the P.sup.-5 side chain acted as a single-site TIP-1
affinity switch.
[0121] Compared to the >1500-fold loss of affinity achieved by a
Trp/Leu substitution in iCAL36, a P.sup.-5 Trp/Ala substitution in
the .beta.-catenin C-terminus caused only a 100-fold loss of TIP-1
affinity (Zhang, et al. (2008) supra). The greater sensitivity of
the iCAL36 sequence could be due to the orientation of its Trp side
chain within the TIP-1 binding pocket, which differs from that
observed in the TIP-1:.beta.-catenin complex (Zhang, et al. (2008)
supra). Alternatively, the differential free-energy change could be
due to the different replacement side-chains (Ala vs. Leu). In
particular, analysis of the TIP-1 P.sup.-5 pocket suggests that it
could not readily accommodate the larger branched. Leu side chain
at this position. To determine the relative contributions of Trp
affinity and/or Leu incompatibility to the iCAL42 binding energy, a
P.sup.-5 alanine mutant of iCAL36 was synthesized and its binding
was tested by FP displacement. The ANSRAPTSII sequence (SEQ ID
NO:22) exhibited a similar lack of affinity for TIP-1 as did
iCAL42. Thus, it appeared that the thermodynamic impact of the
P.sup.-5 substitution on the TIP-1:iCAL36 interaction primarily
reflected the loss of the Trp side chain in stabilizing this
complex, rather than a specific incompatibility of Leu.
[0122] iCAL42 is a Single-PDZ Inhibitor of Endogenous CAL.
[0123] Exploiting the localized vulnerability of the TIP-1 binding
site for iCAL36, a dramatic increase in inhibitor selectivity
against known off-target interactions was generated, as measured by
the difference between the free energy of a given peptide binding
to the CAL PDZ domain and the free energy of the same peptide
binding to the highest affinity alternative among the NHERF and
TIP-1 PDZ domains (.DELTA.G). The SSR5 starting sequence bound CAL
almost exactly as tightly as the closest NHERF1 or NHERF2 domain,
N2P2 (.DELTA..DELTA.GCAL-best=+0.1 kcal/mol). While the binding
free energy of iCAL36 for CAL was much more favorable than for the
NHERF PDZ domains (.DELTA..DELTA.G=-3.3 kcal/mol), it was actually
1.0 kcal/mole less favorable than for TIP-1 (.DELTA..DELTA.G=+1.0
kcal/mol). iCAL42 reversed this trend, binding CAL with a free
energy that was substantially more favorable than any of the other
partners (.DELTA..DELTA.G=-2.5 kcal/mol). Thus, the reward for a
five-fold reduction in CAL binding affinity was a 60-fold
difference relative to the K.sub.i of the PDZ domain with the next
highest affinity.
[0124] To validate these observations for full-length proteins in
the presence of potential physiological accessory proteins, a WCL
pull-down assay was used, together with a biotinylated analog of
iCAL42, BT-iCAL42. The FP competition assay was used to ensure that
the selectivity profile was not compromised by the addition the
N-terminal biotin linker. As expected, BT-iCAL42 bound CAL robustly
(K.sub.i=9.2 .mu.M), but exhibited no appreciable binding for the
NHERF and TIP-1 PDZ domains. In a WCL pull-down immunoassay,
BT-iCAL42 was used as bait, and captured prey proteins were eluted
by displacement with unlabeled iCAL42. When probed by western blot
analysis, full-length CAL was clearly identified, but neither
NHERF1, NHERF2, NHERF3, nor TIP-1 were observed.
[0125] To assess the possibility that the Trp.fwdarw.Leu
substitution might have generated unanticipated off-target
interactions, in analogy to that originally seen for iCAL36 with
TIP-1, the BT-iCAL42 pull-down assay was repeated and putative
interactors were resolved by TCA precipitation, SDS-PAGE, and
silver staining. Aside from a modest enrichment of CAL, no protein
bands were enriched in the iCAL42 eluate compared to the
scrambled-peptide control eluate; nevertheless, all major bands
were submitted for mass-spectrometric analysis. Consistent with
western blot analysis, endogenous CAL was again clearly identified.
Moreover, when the stringency of the pull-down assay was reduced,
there were no other PDZ-domain containing protein in the eluate.
Based on these data, among the PDZ proteins expressed in
CFBE-.DELTA.F epithelial cells, CAL was the only one with
appreciable affinity for iCAL42.
[0126] F*-iCAL42 Enhances CFTR-Mediated Cl.sup.- Secretion.
[0127] The strict selectivity of iCAL42 was further used to test
whether the off-target TIP-1 interaction might contribute to the
.DELTA.F508-CFTR rescue seen with iCAL36. For these studies, the
enhanced CAL selectivity of decapeptides carrying an N-terminal
fluorescein moiety was exploited. For TIP-1, the affinity of
F*-iCAL36 was only three-fold stronger than that of unlabeled
iCAL36, compared to a 13-fold increase for CAL. Therefore, an
N-terminally fluoresceinated version of iCAL42 (F*-iCAL42) was
synthesized and binding against both CAL and TIP-1 was analyzed. In
the context of the iCAL42 sequence, the addition of the N-terminal
fluorescein moiety produced a five-fold enhancement in CAL
affinity. Conversely, the fluoresceinated peptide showed no
appreciable binding to TIP-1: at the highest protein concentration
tested (150 .mu.M), F*-iCAL42 was essentially indistinguishable
from a fluoresceinated scrambled control peptide F*-SCR.
[0128] Having validated the affinity profile of the fluoresceinated
probe, it was determined whether F*-iCAL42 would be able to rescue
.DELTA.F508-CFTR chloride-channel activity as efficiently as
F*-iCAL36. In Ussing chamber measurements, F*-iCAL36 and F*-iCAL42
were tested in head-to-head measurements for efficacy versus the
scrambled control peptide, F*-SCR. The results of this analysis
indicated that F*-iCAL36 increased the CFTR.sub.inh-172-sensitive
short-circuit current (.DELTA.I.sub.sc) by 10.7% (p=0.0016; n=10).
Treatment of CFBE-.DELTA.F cells with F*-iCAL42 yielded a 12.5%
increase (p=0.0013; n=10) in .DELTA.I.sub.sc. Thus, F*-iCAL42 was
at least as efficacious as F*-iCAL36, suggesting that TIP-1
inhibition was not a substantial component of iCAL-mediated
chloride-channel rescue.
Example 5
A Computationally Designed PDZ Domain Peptide Inhibitor Rescues
CFTR Activity
[0129] Retrospective Validation of the K* Algorithm.
[0130] K* predictions were made for peptide sequences from a CAL
peptide-array. The peptide-array binding data were used to validate
the peptide inhibitor predictions. The resulting receiver operating
curve (ROC) when comparing the K* scores to the CAL binding of the
peptide array had an area under the curve (AUC) of 0.84, which
showed that K* greatly enriched for peptides that bind CAL.
[0131] Considering if a prospective test were being conducted and
the top 30 K*-ranked sequences were being tested, according to the
peptide array, 11 of the top 30 sequences would be found to bind
CAL. Notably, this was a 20-fold increase over the number of
binders that would be expected to be found if the binding sequences
were distributed randomly in the rankings.
[0132] Based on previous studies (Reynolds, et al. (2008) J. Mol.
Biol. 382:1265-1275), CAL was known to bind the canonical sequence
motif: X-S/T-X-L/V/I (SEQ ID NO:39). Therefore, a much more
stringent test of the K* design algorithm was to determine the
degree to which K* enriched for binders if the peptide array was
restrict to sequences that matched the known CAL sequence motif.
With this new restriction, K* was still able to significantly
enrich for CAL peptide binders producing a ROC with an AUC of 0.71.
When considering the top 30 K* ranked sequences, 17 of the
sequences were binders, which resulted in a 2-fold increase over
the expected random distribution.
[0133] Prospective Design of CAL Peptide Inhibitors.
[0134] Since K* was able to successfully enrich for CAL binders
based on peptide array data, K* was then used to prospectively find
novel CAL peptide inhibitors. The K* algorithm was used to search
over 2166 possible peptide hexamer inhibitors that had an
N-terminal W-Q pair followed by four residues that matched the CAL
PDZ sequence motif. The top-ranked sequences were chosen to be
experimentally validated. The K.sub.i value for each peptide
hexamer was determined using fluorescence polarization.
[0135] All of the top-ranked inhibitors were novel and none had
been predicted or experimentally tested before. Unexpectedly, all
of the top predicted peptides bound CAL with high affinity
(.DELTA.G.sub.binding in the range of -8 to -6 kcal/mol). The best
binding predicted peptide (kCAL01, WQVTRV; SEQ ID NO:23) had a
K.sub.i of 2.1 .mu.M. For comparison, the K.sub.i for the wild-type
CFTR sequence (TEEEVQDTRL; SEQ ID NO:40) is 690 .mu.M and the
highest known affinity natural ligand (ANGLMQTSKL; SEQ ID NO:38)
for CAL is 37 .mu.M. Using the K* design algorithm, a peptide
inhibitor with 331-fold higher affinity was obtained. Thus, the
design algorithm successfully identified high affinity peptide
inhibitors of the CAL PDZ domain.
[0136] The highest-affinity CAL-binding peptide hexamer (iCAL35,
WQTSII; SEQ ID NO:36) identified through SPOT arrays had a K.sub.i
of 14.8 .mu.M. Seven of the eleven top tested sequences showed an
improvement in binding compared to iCAL35, and kCAL01 showed a
7-fold improvement over iCAL35. The best inhibitor found through
the SPOT array screens involved a fluorescein group modification to
a peptide decamer (F*-iCAL36, F*-ANRSWPTSII (SEQ ID NO:19),
K.sub.d=1.3 .mu.M). kCAL01 rivaled this binding affinity despite
the computational search library restriction to only allow amino
acids and hexamer sequences. Critically, at nearly half the size
(830 Da) of F*-iCAL36, kCAL01 had approximately twice the binding
efficiency (ratio of inhibitor potency to size) of F*-iCAL36 and
was much closer in size to typical drugs.
[0137] Furthermore, the tight binding of the top-ranked sequences
was not merely a consequence of the underlying CAL-binding motif
used to select candidate sequences for evaluation. To confirm this,
a set of poorly-ranked sequences was synthesized and their
CAL-binding affinity was experimentally evaluated. Almost all of
the poorly-ranked sequences bound CAL, consistent with their
motifs. Reflecting the enrichment of CAL binders in the pool, the
two poorly ranked peptides with the highest affinities
(K.sub.i=.mu.M and 27 .mu.M, respectively) were indeed close to the
affinity of the weakest top-ranked sequence (K.sub.i=18 .mu.M).
However, all of the poorly ranked peptides bound CAL more weakly
than any of the top-ranked sequences, and none of them had improved
affinity relative to prior biochemical efforts. Thus, K* was a
powerful filter, efficiently selecting tight binders from a pool of
sequences with baseline affinity for the target.
[0138] Biological Activity of the Best Designed Peptide
Inhibitor.
[0139] All of the top-predicted inhibitors successfully bound CAL.
This implied that the inhibitors could disrupt the degradation
pathway of CFTR. However, to restore CFTR function in epithelial
cells, the inhibitor must be specific for CAL and not bind other
CFTR trafficking proteins. Interestingly, the top-binding predicted
peptide contained a .beta.-branched C-terminal residue (Val) that
was preferred by CAL, but not by NHERF PDZ domains.
[0140] The ability of the top designed peptide, kCAL01, to restore
.DELTA.F508-CFTR function was determined by measuring
.DELTA.F508-CFTR-mediated chloride efflux in cystic fibrosis
patient-derived bronchial cells expressing .DELTA.F508-CFTR
(CFBE-.DELTA.F) using an Ussing chamber. This analysis compared
.DELTA.F508-CFTR chloride flux for a control peptide (kCAL31;
WQDSGI (SEQ ID NO:41); no CAL binding expected), iCAL35, and
kCAL01. While there was only a slight improvement in chloride flux
for iCAL35 over the control peptide (4%), the designed peptide
kCAL01 exhibited a much larger increase (12%). The 12% increase in
.DELTA.F508-CFTR chloride efflux was similar to the rescue of
activity when using the selective peptide F*-iCAL36. Thus, the
designed peptide kCAL01 was biologically active and of use in
inhibiting the interaction between CAL and CFTR.
Example 6
iCAL Peptide Boosts Functional Rescue in Combination with a
Corrector
[0141] To assess the potential for complementary action between a
CAL inhibitor and a corrector, the inhibitor of CAL peptide iCAL36
(SEQ ID NO:20) was analyzed in combination with corr-4a. CF patient
airway epithelial cells expressing .DELTA.F508-CFTR (CFBE-.DELTA.F
cells) were treated with either corr-4A or DMSO and either
F*-iCAL36 (fluoresceinated peptide) or a scrambled control peptide
(F*-SCR). Following treatment with corr-4a alone, CFBE-.DELTA.F
monolayers showed levels of chloride-channel activity .about.15%
higher than the control. However, when treated with corr-4a and
iCAL36, CFBE-.DELTA.F monolayers showed .about.25% increase
(Cushing, et al. (2010) Angewandte 49:9910). Furthermore, CAL
knockdown can enhance apical levels of even wild-type CFTR by
>2-fold, indicating that even corrected channels can be
stabilized by CAL inhibitors.
Example 7
Bioavailable CAL Inhibitor and Combination Therapy
[0142] The inhibitor of CAL peptide iCAL36 (SEQ ID NO:20) blocks
the CAL PDZ binding site, extends CFTR apical half-life, and
increases .DELTA.F508-CFTR chloride currents alone and in concert
with a CFTR corrector. To evaluate the therapeutic potential of a
bioavailable CAL inhibitor and to test a key peptide modification
strategy, the iCAL36 N-terminus was functionalized with the
cell-penetrating peptide MPG (GALFLGFLGAAGSTMGAWSQPKKKRKV; SEQ ID
NO:42; Morris, et al. (1997) Nucleic Acids Res 25:2730-2736),
yielding miCAL36. Using this bioavailable CAL inhibitor in
combination with a CFTR corrector such as Lumacaftor and a CFTR
potentiator such as Ivacaftor, it is contemplated that all three
defects (folding, gating and stability) of .DELTA.F508-CFTR can be
addressed.
Sequence CWU 1
1
4916PRTArtificial SequenceSynthetic peptide 1Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 211PRTArtificial SequenceSynthetic peptide 2Cys Ala Asn Gly
Leu Met Gln Thr Ser Lys Ile 1 5 10 39PRTArtificial
SequenceSynthetic peptide 3Cys Gly Leu Met Gln Thr Ser Lys Ile 1 5
47PRTArtificial SequenceSynthetic peptide 4Cys Phe Phe Ser Thr Ile
Ile 1 5 57PRTArtificial SequenceSynthetic peptide 5Cys Phe Phe Thr
Ser Ile Ile 1 5 67PRTArtificial SequenceSynthetic peptide 6Cys Met
Gln Thr Ser Ile Ile 1 5 77PRTArtificial SequenceSynthetic peptide
7Cys Met Gln Thr Ser Lys Ile 1 5 87PRTArtificial SequenceSynthetic
peptide 8Cys Trp Gln Thr Ser Ile Ile 1 5 97PRTArtificial
SequenceSynthetic peptide 9Cys Trp Pro Thr Ser Ile Ile 1 5
108PRTArtificial SequenceSynthetic peptide 10Cys Thr Trp Gln Thr
Ser Ile Ile 1 5 118PRTArtificial SequenceSynthetic peptide 11Cys
Lys Trp Gln Thr Ser Ile Ile 1 5 128PRTArtificial SequenceSynthetic
peptide 12Pro His Trp Gln Thr Ser Ile Ile 1 5 138PRTArtificial
SequenceSynthetic peptide 13Phe His Trp Gln Thr Ser Ile Ile 1 5
148PRTArtificial SequenceSynthetic peptide 14Ser Arg Trp Gln Thr
Ser Ile Ile 1 5 1511PRTArtificial SequenceSynthetic peptide 15Cys
Ala Asn Ser Arg Trp Gln Thr Ser Ile Ile 1 5 10 168PRTArtificial
SequenceSynthetic peptide 16Gly Leu Trp Pro Thr Ser Ile Ile 1 5
178PRTArtificial SequenceSynthetic peptide 17Ser Arg Trp Pro Thr
Ser Ile Ile 1 5 188PRTArtificial SequenceSynthetic peptide 18Phe
Pro Trp Pro Thr Ser Ile Ile 1 5 1910PRTArtificial SequenceSynthetic
peptide 19Ala Asn Ser Arg Trp Pro Thr Ser Ile Ile 1 5 10
2010PRTArtificial SequenceSynthetic peptide 20Ala Asn Ser Arg Trp
Pro Thr Ser Ile Ile 1 5 10 2110PRTArtificial sequenceSynthetic
peptide 21Ala Asn Ser Arg Leu Pro Thr Ser Ile Ile 1 5 10
2210PRTArtificial sequenceSynthetic peptide 22Ala Asn Ser Arg Ala
Pro Thr Ser Ile Ile 1 5 10 236PRTArtificial sequenceSynthetic
peptide 23Trp Gln Val Thr Arg Val 1 5 246PRTArtificial
SequenceSynthetic peptide 24Xaa Xaa Xaa Xaa Xaa Xaa 1 5
2515PRTArtificial sequenceSynthetic peptide 25Trp Arg Phe Lys Lys
Ala Asn Ser Arg Trp Pro Thr Ser Ile Ile 1 5 10 15 2615PRTArtificial
sequenceSynthetic peptide 26Trp Arg Phe Lys Lys Ala Asn Ser Arg Trp
Pro Thr Ser Ile Ile 1 5 10 15 2715PRTArtificial sequenceSynthetic
peptide 27Trp Arg Phe Lys Lys Ala Asn Ser Arg Trp Pro Thr Ser Ile
Ile 1 5 10 15 2810PRTArtificial sequenceSynthetic peptide. 28Pro
Asn Glu Ala Trp Pro Thr Ser Ile Ile 1 5 10 2910PRTArtificial
sequenceSynthetic peptide. 29Phe Asn Ala Arg Trp Gln Thr Ser Ile
Ile 1 5 10 3010PRTArtificial sequenceSynthetic peptide 30Phe Asn
Ser Arg Trp Gln Thr Ser Ile Ile 1 5 10 3110PRTArtificial
sequenceSynthetic peptide 31Lys Asn Ser Arg Trp Gln Thr Ser Ile Ile
1 5 10 3210PRTArtificial sequenceSynthetic peptide 32Pro Asn Ser
Arg Trp Gln Thr Ser Ile Ile 1 5 10 3310PRTArtificial
sequenceSynthetic peptide 33Ala Asn Ser Arg Trp Gln Thr Ser Ile Ile
1 5 10 345PRTArtificial sequenceSynthetic peptide 34Trp Arg Phe Lys
Lys 1 5 357PRTArtificial sequenceSynthetic peptide 35Leu Glu Val
Leu Phe Gln Gly 1 5 366PRTArtificial sequenceSynthetic peptide
36Trp Gln Thr Ser Ile Ile 1 5 374PRTArtificial sequenceSynthetic
peptide 37Thr Ser Ile Ile 1 3810PRTArtificial sequenceSynthetic
peptide 38Ala Asn Gly Leu Met Gln Thr Ser Lys Leu 1 5 10
394PRTArtificial sequenceSynthetic peptide 39Xaa Xaa Xaa Xaa 1
4010PRTArtificial sequenceSynthetice peptide 40Thr Glu Glu Glu Val
Gln Asp Thr Arg Leu 1 5 10 416PRTArtificial sequenceSynthetic
peptide 41Trp Gln Asp Ser Gly Ile 1 5 4227PRTArtificial
sequenceSynthetic peptide 42Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala
Ala Gly Ser Thr Met Gly 1 5 10 15 Ala Trp Ser Gln Pro Lys Lys Lys
Arg Lys Val 20 25 438PRTArtificial sequenceSynthetic peptide 43Arg
Arg Arg Arg Arg Arg Arg Arg 1 5 4414PRTArtificial sequenceSynthetic
peptide 44Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gln 1
5 10 4527PRTArtificial sequenceSynthetic peptide 45Gly Trp Thr Leu
Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5 10 15 Lys Ala
Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 25 4621PRTArtificial
sequenceSynthetic peptide 46Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu
Lys Ala Leu Ala Ala Leu 1 5 10 15 Ala Lys Lys Ile Leu 20
4718PRTArtificial sequenceSynthetic peptide 47Lys Leu Ala Leu Lys
Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Ala
4827PRTArtificial sequenceSynthetic peptide 48Gly Ala Leu Phe Leu
Ala Phe Leu Ala Ala Ala Leu Ser Leu Met Gly 1 5 10 15 Leu Trp Ser
Gln Pro Lys Lys Lys Arg Lys Val 20 25 4916PRTArtificial
sequenceSynthetic peptide 49Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg
Arg Met Lys Trp Lys Lys 1 5 10 15
* * * * *